In Situ

General Introduction to In Situ H ybridization
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G eneral Introduction to In Situ Hybridization

I n situ hybridization techniques allow specific nucleic acid sequences to be detected in morphologically preserved chromosomes, cells or tissue sections. In combination with immunocytochemistry, in situ hybridization can relate microscopic topological information to gene activity at the D N A, mRN A, and protein level. The technique was originally developed by Pardue and G all (1969) and (independently) by John et al. (1969). At this time radioisotopes were the only labels available for nucleic acids, and autoradiography was the only means of detecting hybridized sequences. Furthermore, as molecular cloning was not possible in those days, in situ hybridization was restricted to those sequences that could be purified and isolated by conventional biochemical methods (e.g., mouse satellite DN A, viral DN A, ribosomal RN As). Molecular cloning of nucleic acids and improved radiolabeling techniques have changed this picture dramatically. For example, D N A sequences a few hundred base pairs long can be detected in metaphase chromosomes by autoradiography (H arper et al., 1981; Jhanwag et al., 1984; Rabin et al., 1984; Schroeder et al., 1984). Also radioactive in situ techniques can detect low copy number mRN A molecules in individual cells (H arper et al., 1986). Some years ago, chemically synthesized, radioactively labeled oligonucleotides began to be used, especially for in situ mRN A detection (Coghlan et al., 1985). In spite of the high sensitivity and wide applicability of in situ hybridization techniques, their use has been limited to research laboratories. This is probably due to the problems associated with radioactive probes, such as the safety measures required, limited shelf life, and extensive time required for autoradiography. In addition, the scatter inherent in radioactive decay limits the spatial resolution of the technique. H owever, preparing nucleic acid probes with a stable nonradioactive label removes the major obstacles which hinder the general application of in situ hybridization. Furthermore, it opens new opportunities for combining different labels in one experiment. The many sensitive antibody detection systems available for such probes further enhances the flexibility of this method. In this manual, therefore, we describe nonradioactive alternatives for in situ hybridization.
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G eneral I ntroduction to I n Situ H ybridiz ation
Direct and indirect methods

There are two types of nonradioactive hybridization methods: direct and indirect. In the direct method, the detectable molecule (reporter) is bound directly to the nucleic acid probe so that probe-target hybrids can be visualized under a microscope immediately after the hybridization reaction. For such methods it is essential that the probe-reporter bond survives the rather harsh hybridization and washing conditions. Perhaps more important, however, is, that the reporter molecule does not interfere with the hybridization reaction. The terminal fluorochrome labeling procedure of RN A probes developed by Bauman et al. (1980, 1984), and the direct enzyme labeling procedure of nucleic acids described by Renz and Kurz (1984) meet these criteria. Boehringer Mannheim has introduced several fluorochrome-labeled nucleotides that can be used for labeling and direct detection of DN A or RN A probes. If antibodies against the reporter molecules are available, direct methods may also be converted to indirect immunochemical amplification methods (Bauman et al., 1981; Lansdorp et al., 1984; Pinkel et al., 1986). Indirect procedures require the probe to contain a reporter molecule, introduced chemically or enzymatically, that can be detected by affinity cytochemistry. Again, the presence of the label should not interfere with the hybridization reaction or the stability of the resulting hybrid. The reporter molecule should, however, be accessible to antibodies. A number of such hapten modifications has been described (Langer et al., 1981; Leary et al., 1983; Landegent et al., 1984; Tchen et al., 1984; H opman et al., 1986; H opman et al., 1987; Shroyer and N akane, 1983; Van Prooijen et al., 1982; Viscidi et al., 1986; Rudkin and Stollar, 1977; Raap et al., 1989). O ne of the most popular is the biotin-streptavidin system. Boehringer Mannheim offers an alternative, the Digoxigenin (DIG/Genius) System which is described in detail later in this chapter. A few years ago, the chemical synthesis of oligonucleotides containing functional groups (e.g., primary aliphatic amines or sulfhydryl groups) was described. These can react with haptens, fluorochromes or enzymes to produce a stable probe which can be used for in situ hybridization experiments (Agrawal et al., 1986; C hollet and Kawashima, 1985; H aralambidis et al., 1987; Jablonski et al., 1986). Modified oligonucleotides can also be obtained with the D IG / G enius system (Mhlegger et al., 1990). Such oligonucleotide probes will undoubtedly be widely used as automated oligonucleotide synthesis makes them available to researchers not familiar with D N A recombinant technology. This manual concentrates on two labeling systems: Indirect methods using digoxigenin (detected by specific antibodies) and biotin (detected by streptavidin) D irect methods using fluorescein or other fluorochromes directly coupled to the nucleotide The ordering information in C hapter 7 lists all the kits and single reagents Boehringer Mannheim offers for nonradioactive labeling and detection.
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Guidelines for In Situ H ybridization
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G uidelines for In Situ Hybridization

An in situ hybridization protocol follows this general outline: Preparation of slides and fixation of material Pretreatments of material on slides, e.g., permeabilization of cells and tissues D enaturation of in situ target D N A (not necessary for mRN A target) Preparation of probe I n situ hybridization Posthybridization washes Immunocytochemistry Microscopy All these steps are discussed below in detail. The information provided will make it easier to decide whether a certain step should or should not be included in a given in situ hybridization protocol.
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Details of the Technique

Slide preparation
For chromosome spreads, alcohol/ ether (1:1) cleaned slides are sufficient. H owever, since tissue sections may be lost during the procedure, use either polylysine or glutaraldehyde-activated gelatin chrome aluminum slides for these sections.
Pretreatments of material on the slide

Endogenous enzyme inactivation
Fixation
To preserve morphology, the biological material must be fixed. From a chemical point of view, there is little limitation in the type of fixation used because one of the following will be true: The functional groups involved in base pairing are protected in the double helix structure of duplex D N A. RN A is fairly unreactive to crosslinking agents. The reaction is reversible (e.g., with formaldehyde). For metaphase chromosome spreads, methanol/acetic acid fixation is usually sufficient. For paraffin-embedded tissue sections, use formalin fixation. Cryostat sections fixed for 30 min with 4% formaldehyde or with Bouins fixative have been used successfully, as well as paraformaldehyde vapor fixation. Tissues can also be freeze-dried. It should be noted that the D N A and RN A target sequences are surrounded by proteins and that extensive crosslinking of these proteins masks the target nucleic acid. Therefore, permeabilization procedures are often required. U nfortunately, a fixation protocol which can be used for all substrates has not yet been described. The fixation and pretreatment protocols must be optimized for different applications.
When an enzyme is used as the label, the endogenous enzyme activity may have to be inactivated. For peroxidase, this is done by treating the sections with 1% H 2O 2 in methanol for 30 min. For alkaline phosphatase, levamisole may be added to the substrate solution, but this may be unnecessary since residual alkaline phosphatase activity is usually lost during hybridization.
RNase treatment
RN ase treatment serves to remove endogenous RN A and may improve the signal-tonoise ratio in DN A-DN A hybridizations. This treatment can also be used as a control in hybridizations with (m)RN A as target. It is done by incubating the preparations in DN ase-free RN ase (100 g/ml) in 2 x SSC at 37C for 60 min. (SSC = 150 mM N aCl, 15 mM sodium citrate, pH 7.4).
HCl treatment
In several protocols a 2030 min treatment with 200 mM H C l is included. The precise action of the acid is not known, but extraction of proteins and partial hydrolysis of the target sequences may contribute to an improvement in the signal-to-noise ratio.
Detergent treatment
Preparations may be pretreated with Triton X-100, sodium dodecyl sulfate, or other detergents if lipid membrane components have not been extracted by other procedures such as fixation, dehydration, embedding, and endogenous enzyme inactivation procedures.
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D etails of the Technique
Protease treatment
Denaturation of probe and target

For in situ hybridization to chromosomal D N A, the D N A target must be denatured. In general such treatments may lead to loss of morphology, so in practice, a compromise must be found between hybridization signal and morphology. Alkaline denaturations have traditionally been used. H eat denaturations have also become popular, because of their experimental simplicity and greater effectiveness. Variations in time and temperature should be evaluated to find the best conditions for denaturation. For heat denaturation, the probe and target chromosomal D N A may be denatured simultaneously. To accomplish this, put the probe on the slide and cover with a coverslip. Bring the slide to 80C for 2 min in an oven for the denaturation and then cool to 37C . For tissue sections, if necessary, extend the time of denaturation at 80C to 10 min. For competition in situ hybridization, where the labeled probe is allowed to reanneal with unlabeled competitor D N A, denature the chromosomal preparation and probe separately.
Protease treatment serves to increase target accessibility by digesting the protein that surrounds the target nucleic acid. To digest the sample, incubate the preparations with up to 500 g/ ml Proteinase K (the optimal amount must be determined) in 20 mM Tris-H C l, 2 mM C aC l2, pH 7.4, for 7.530 min at 37C . For example, with chromosomes or isolated preparations of nuclei, a reasonable starting point for protease digestion is up to 1 g/ ml Proteinase K for 7.5 min. For formalin-fixed material, 515 g/ ml for 1530 min usually gives good results. The use of pepsin has been shown to give excellent results for formalin-fixed, paraffinembedded tissue sections. Routine pepsin digestion involves incubating the preparations for 30 min at 37C in 200 mM H C l containing 500 g/ ml pepsin. For the pretreatment of chromosome spreads with pepsin see page 70. O ther hydrolases, such as C ollagenase and diastase, may be tried if preparations of connective tissue and liver give high background reactions. Also, freeze/ thaw cycles have been used to improve the penetration of probes into tissues.
Hybridization
See C hapter 3 for the composition of the hybridization solution, and the factors affecting hybridization kinetics and hybrid stability.
Prehybridization
A prehybridization incubation is often necessary to prevent background staining. The prehybridization mixture contains all components of a hybridization mixture except for probe and dextran sulfate.
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Posthybridization washes
Labeled probe can hybridize nonspecifically to sequences which are partially but not entirely homologous to the probe sequence. Such hybrids are less stable than perfectly matched hybrids. They can be dissociated by performing washes of various stringencies (as described in Chapter 3). The stringency of the washes can be manipulated by varying the formamide concentration, salt concentration, and temperature. O ften a wash in 2 x SSC containing 50% formamide suffices. For some applications the stringency of the washes should be higher. H owever, we recommend hybridizing stringently rather than washing stringently.
Fluorescence
Useful fluorochromes for fluorescence in situ hybridization (FISH ) include the blue fluorochrome AMCA (amino-methylcoumarin-acetic acid), the green fluorophore fluorescein, and the red fluorochromes CY3, rhodamine, and Texas Red. Recently, the FISH application of an infrared dye has been reported (Ried et al., 1992), although it can only be visualized with infrared-sensitive cameras. For routine experiments, several immunofluorophores with good spectral separation properties can be used for FISH analysis (Table 1). Chapter 5 contains detailed procedures for using all these fluorochromes. Color AMC A Fluorescein C Y3 Rhodamine Texas Red Blue G reen Red Red Red Excitation max. (nm) 399 494 552 555 590
2
Emission max. (nm) 446 523 565 580 615
Table 1: Spectral properties of fluorophores used in FISHanalysis.
Immunocytochemistry
In this manual immunocytochemical procedures using digoxigenin, biotin, and fluorochromes are described. These allow flexibility in the choice of the final reporter molecule and type of microscopy.
Blocking reaction
U se a blocking step prior to the immunological procedure to remove high background. For example, perform biotin detection steps in PBS containing Tween 20 and BSA (when using monoclonal antisera) or normal serum (when using polyclonal antisera). Perform routine digoxigenin detection in Tris-H C l buffer containing Blocking Reagent (instead of BSA or normal serum) to remove background. Also the addition of 0.4 M N aC l can help prevent background staining if the antigen/ hapten bond can survive the high salt conditions. In a standard reaction, block the target tissue, cell, or chromosome preparation and then incubate it with the antibodyconjugate solution for 30 min at 37C (or 2 h at room temperature) in a moist chamber. Afterwards, wash the slide three times (510 min each) with buffer containing Tween 20.
Rhodamine-, fluorescein-, and coumarinbased dyes cover the three primary colors of the visible part of the electro-magnetic spectrum. By using selective excitation and emission filters, and dichroic mirrors, these colors can be visualized without crosstalk, making triple color FISH feasible. To increase the identification of targets by color, a combinatorial labeling approach has been developed (N iederlof et al., 1990; Ried et al., 1992; Wiegant et al., 1993). Multiplicity is then given by 2n 1, where n is the number of colors. After establishing that hybridized probes labeled with two haptens in different ratios still fluoresce at fairly constant ratios, laboratories extended the combinatorial approach to ratio-labeling (N ederlof et al., 1992). In ratio-labeling, the ratio of fluorescence intensities of the various color combinations is used for color identification of the probes. This approach allowed simultaneous identification of twelve different probes (D auwerse et al., 1992). For detailed information, refer to the literature describing combinatorial and ratio labeling (Wiegant and D auwerse, 1995).
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Fluorescent DN A counterstaining is usually performed with red fluorescent propidium iodide (PI) or with blue fluorescent DAPI. Currently, new counterstains are being introduced, e.g., green fluorescent YO YO -1 (Molecular Probes, Inc., USA).
An antifading agent should be added to the embedding medium to retard fading. When fluorescent labels are used, the recommended antifading agent is Vectashield (Vector Laboratories). The different D N A counterstains can be directly dissolved in the embedding medium (40 ng D API/ ml; 100 ng PI/ ml; or 0.1 M YO YO -1/ ml) or the fluorescent specimen can first be counterstained and then embedded in Vectashield. If desired, the coverslip may be sealed with nail varnish.
Enzymes
When using brightfield microscopy with peroxidase, prepare the following peroxidase substrate solution: 0.5 mg/ ml diaminobenzidine in 50 mM Tris-H C l, pH 7.4, containing 10 mM imidazole. Shortly before use, add 0.05% H 2O 2. Incubate with substrate from 230 min (depending on the signal-to-noise ratio). We advise inspecting sections microscopically during the peroxidase reaction. Stop the reaction by washing the sample with water. C ounterstain with G iemsa if desired. In the case of reflection contrast microscopy with peroxidase, prepare the following peroxidase substrate solution: 0.1 mg/ ml diaminobenzidine in 50 mM Tris-H C l, pH 7.4. Shortly before use, add 0.01% H 2O 2. The usual reaction time is 10 min. After stopping the reaction with H 2O and dehydrating with ethanol, evaluate slides by oil-immersion microscopy without embedding. When using Alkaline Phosphatase, prepare the following substrate: 0.16 mg/ml 5-Bromo4-chloro-3-indolyl-phosphate (BC IP) and 0.33 mg/ ml N itro-blue tetrazolium salt (N BT) in 200 mM Tris-H C l, 10 mM MgC l2, pH 9.2. D etermine reaction times by evaluating the signal-to-noise ratio, which should be checked microscopically during the enzyme reaction. The reaction medium itself is stable (in the dark). The final product (N BT formazan) also has reflective properties. A recently introduced alkaline phosphatase substrate (H N PP/ Fast Red TR) also allows fluorescent detection of alkaline phosphatase label. For fluorescence microscopy, prepare the following substrate: 10 mg/ ml H N PP (in dimethylformamide) and 25 mg/ ml Fast Red TR in redist H 2O . For details on the use of H N PP/ Fast Red TR, see the article in C hapter 5, page 108.
U se any enzyme commonly used in immunocytochemistry. We show examples for peroxidase and alkaline phosphatase. With peroxidase (PO D), use the diaminobenzidine (DAB)/imidazole reaction (Graham and Karnovsky, 1966). With alkaline phosphatase (AP), use the 5-Bromo-4-chloro-3indolylphosphate/N itro-blue tetrazolium (BCIP/N BT) reaction. The advantages of these colorimetric detection methods are good localization properties, high sensitivity (De Jong et al., 1985; Straus, 1982; Scopsi and Larson, 1986) and stability of the precipitates. As the D AB reaction produces a color which contrasts with the blue alkaline phosphatase staining reaction (and vice versa), one can use both enzymes in double hybridizations. Furthermore, both detection methods have bright reflective properties. They can be amplified by gold/ silver (G allyas et al., 1982; Burns et al., 1985) and the color can be modified with heavy metal ions (H su and Soban, 1982; C remers et al., 1987).
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Microscopy
Brightfield microscopy
In brightfield microscopy the image is obtained by the direct transmission of light through the sample. Evaluation of in situ hybridization results by brightfield microscopy is preferred for most routine applications because the preparations are permanent. H owever, the sensitivity demanded by many applications (e.g., single copy gene localization requires the detection of a few attograms of D N A) may require more sophisticated microscopy.
Darkfield microscopy
al., 1985a) the DAB/peroxidase product displays bright reflection when present in extremely low amounts (i.e., low local absorption, typically A < 0.05). This property has made reflection contrast microscopy instrumental in detecting the first single copy gene by nonradioactive means (Landegent et al., 1985b; H opman et al., 1986b; Ambros et al., 1986). Studies of DAB image formation in reflection contrast microscopy have shown that best results require thin objects and low local absorbances of the DAB product (Cornelese ten Velde et al., 1988).
Fluorescence microscopy
In the darkfield microscope, the illuminating rays of light are directed from the side, so that only scattered light enters the microscope lenses. C onsequently, the material appears as an illuminated object against a black background. D arkfield microscopy is extensively used for radioactive in situ hybridization experiments because large fields can be examined at low magnification. The distribution of the silver grains can be seen with high contrast. In contrast, these types of microscopy are seldom used to localize reaction products of nonradioactive hybridization procedures (H eyting et al., 1985). H owever, Garson et al. (1987) shows the application of phase contrast microscopy to single copy gene detection.
Phase contrast microscopy
A fluorescence microscope contains a lamp for excitation of the fluorescent dye and a special filter which transmits a high percentage of light emitted by the fluorescent dye. Usually antifading reagents have to be added before analysis. Fluorescence microscopy is of great value for nonradioactive in situ hybridization. It is highly sensitive. Furthermore, it can be used to excite three different immunofluorophores with spectrally well-separated emissions (which allows multiple detection). Also the option of using highly sensitive image acquisition systems and the relative ease of quantitation of fluorescence signals adds to its attractiveness.
Digital imaging microscopy
Phase contrast microscopy exploits the interference effects produced when two sets of waves combine. This is the case when light passing e.g., through a relatively thick or dense part of the cell (such as the nucleus) is retarded and its phase consequently shifted relative to light that has passed through an adjacent (thinner) region of the cytoplasm. Phase contrast microscopy is often used for enzyme detection.
Reflection contrast microscopy
Digital imaging microscopes can detect signals that cannot be seen with conventional microscopes. Also, image processing technology provides enhancement of signal-tonoise ratios as well as measurement of quantitative data. The most sensitive system to date, the cooled CCD (charged coupled device) camera, counts emitted photons with a high efficiency over a broad spectral range of wavelengths, and thus is the instrument of choice for two-dimensional analysis. For three-dimensional analysis, confocal laser scanning microscopy is used.
Electron microscopy
Reflection contrast microscopy is similar to phase contrast microscopy. This technique measures the shift of wavelength of the light reflected from the sample relative to that of directly emitted light. Bonnet (personal communication) made the crucial observation that with reflection contrast microscopy (Ploem, 1975; Van der Ploeg and Van D uijn, 1979; Landegent et
The resolution of microscopy is enhanced when electrons instead of light are used, since electrons have a much shorter wavelength (0.004 nm). The practical resolving power of most modern electron microscopes is 0.1 nm. With the electron microscope, the fine structure of a cell can be resolved. Such analysis requires special preparative procedures which are described in detail in Chapter 5.
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Flow cytometry
The enormous speed with which fluorescence of individual cells can be measured with a flow cytometer has many advantages for quantitating in situ hybridization signals. Procedures for fluorescence in situ hybridization in suspension have been described (Trask et al., 1985) and further developments in this field will be very important.
Flow diagram for in situ hybridization

Preparation of slides/ coverslips e.g., gelatin or poly-lysine treatment of slides siliconization of coverslips Fixation of material on slide by precipitation (e.g., ethanol) by cross-linkage (e.g., formaldehyde) Pretreatment of specimen (optional) a) Treatments to prevent background staining endogenous enzyme inactivation RNase-treatment b) Permeabilization diluted acids detergent/alcohol proteases Prehybridization (optional) Incubation of specimen with a pre-hybridization solution (= hybridization solution minus probe) is performed at the same temperature as hybridization Denaturation of probe and target pH or heat simultaneous or separate denaturation of probe and target (if double stranded) Hybridization Components of the solution are mainly: Denhardts Mix (Ficoll, BSA, PVP) heterologous nucleic acids (e.g., herring sperm DNA/ tRNA/competitor DNA) sodium phosphate, EDTA, SDS, salt formamide dextran sulfate Post-hybridization steps treatment with single strand specific nuclease (optional) stringency washes Immunological detection blocking step antibody incubation colorimetric substrate or fluorescence microscopy counterstaining mounting Microscopy microscopic analysis of results and documentation Evaluation Determination of hybridization specificity (controls) nuclease treatment use of multiple probes use of heterologous nucleic acid/vectors
Probe a) Choice of the probe ds: DNA, cDNA ss: RNA, oligonucleotide ss-DNA b) Preparation of the probe DNA: fragment isolation (optional) cDNA: cloning RNA: cloning in transcription vectors oligonucleotides: chemical synthesis c) Labeling of the probe ds DNA: random primed DNA labeling, nick translation, PCR RNA: In vitro transcription, RT-PCR oligonucleotides: endlabeling or tailing Determination of hybridization conditions, e.g., determination of hybridization temperature, pH, use of formamide, salt concentration composition of hybridization solution probe concentration
Fluorescence microscopy for probes directly labeled with a fluorochrome
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References
Ambros, P .F .; Matzke, M. A.; Matzke, A. J. M. (1986) Detection of a 17 kb unique (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94, 1116. Burns, J.; Chan, V .T .W .; Jonasson, J. A.; Fleming, K. A.; Taylor, S.; McG ee, J. O. D. (1985) Sensitive system for visualizing biotinylated DNA probes hybridized in situ: rapid sex determination of intact cells. J. Clin. Pathol. 38, 10851092. Cornelese ten Velde, I.; Bonnet, J.; Tanke, H. J.; Ploem, J. S. (1988) Reflection contrast microscopy. Visualization of (peroxidase generated) diaminobenzidine polymer products and its underlying optical phenomena. Histochemistry 89, 141150. Cremers, A. F . M.; Jansen in de W al, N.; Wiegant, J.; Dirks, R. W .; W eisbeek, P .; Van der Ploeg, M.; Landegent, J. E. (1987) Nonradioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection contrast microscopy and electron microscopy. Histochemistry 86, 609615. Dauwerse, J. G .; Wiegant, J.; Raap, A. K.; Breuning, M. H.; Van Ommen, G . J. B. (1992) Multiple colors by fluorescence in situ hybridization using ratiolabeled DNA probes create a molecular karyotype. Hum. Molec. G enet. 1, 593598. De Jong, A. S. H.; Van Kessel-Van Vark, M.; Raap, A. K. (1985) Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry. Histochem. J. 17, 11191130. G allyas, F .; G orcs, T .; Merchenthaler, I. (1982) High grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J. Histochem. Cytochem. 30, 183184. G arson, J. A.; Van den Berghe, J. A.; Kemshead, J. T . (1987) Novel non-isotopic in situ hybridization technique detects small (1 kb) unique sequences in routinely G -banded human chromosomes: fine mapping of N-myc and b-NG F genes. Nucleic Acids Res. 15, 47614770. G raham, R. C.; Karnovsky, M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291302. Heyting, C.; Kroes, W .G . M.; Kriek, E.; Meyer, I.; Slater, R. (1985) Hybridization of N-acetoxy-N-acetyl2-aminofluorene labeled RNA to Q-banded metaphase chromosomes. Acta Histochem. 77, 177184. Hopman, A. H. N.; Wiegant, J.; Raap, A. K.; Landegent, J. E.; Van der Ploeg, M.; Van Duijn, P . (1986b) Bi-color detection of two target DNAs by nonradioactive in situ hybridization. Histochemistry 85, 14. Hsu, S. M.; Soban, E. (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J. Histochem. Cytochem. 30, 10791082. Landegent, J. E., Jansen in de W al, N.; Ommen, G . J. B.; Baas, F .; De Vijlder, J. J. M.; Van Duijn, P .; Van der Ploeg, M. (1985b) Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization. Nature 317, 175177. Landegent, J. E.; Jansen in de W al, N.; Ploem, J. S.; Van der Ploeg, M. (1985a) Sensitive detection of hybridocytochemical results by means of reflection contrast microscopy. J. Histochem. Cytochem. 33, 12411246. Nederlof, P .M.; Van der Flier, S.; Wiegant, J.; Raap, A. K.; Tanke, H. J.; Ploem, J. S.; Van der Ploeg, M. (1990) Multiple fluorescence in situ hybridization. Cytometry 11, 126131. Nederlof, P . M.; Van der Flier, S.; Vrolijk, J.; Tanke, H. J.; Raap, A. K. (1992) Quantification of in situ hybridization signals by fluorescence digital imaging microscopy. III. Fluorescence ratio measurements of double labeled probes. Cytometry 13, 838845. Ploem, J. S. (1975) Reflection contrast microscopy as a tool for investigation of the attachment of living cells to a glass surface. In: Van Furth, R. (Ed.) Mononuclear Phagocytes in Immunity, Infection and Pathology. Oxford: Blackwell, 405421. Reid, T .; Baldini, A.; Rand, T . C.; W ard, D. C. (1992) Simultaneous visualization of seven different probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. USA 89, 13881392. Scopsi, L.; Larsson, L. I. (1986) Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system. Histochemistry 84, 221230. Straus, W . (1982) Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at a neutral pH. J. Histochem. Cytochem. 30, 491493. Trask, B.; Van den Engh, G .; Landegent, J.; Jansen in de W al, N.; Van der Ploeg, M. (1985) Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry. Science 230, 14011403. Van der Ploeg, M.; Van Duijn, P . (1979) Reflection versus fluorescence. Histochemistry 62, 227232. Wiegant, J.; Wiesmeijer, C. C.; Hoovers, J. M. N.; Schuuring, E.; DAzzo, A.; Vrolijk, J.; Tanke, H. J.; Raap, A. K. (1993) Multiple and sensitive in situ hybridization with rhodamine-, fluorescein-, and enet. 63, coumarin-labeled DNAs. Cytogenet. Cell G 7376. Wiegant, J.; Dauwerse, J. G . (1995) Multiple-colored chromosomes by fluorescence in situ hybridization. In: Verma, R. S.; Babu, A. (Eds.) Human Chromosomes: Principles and Techniques, 2nd ed. New Y ork: McG raw-Hill, Inc.
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Procedures for In Situ H ybridization to Chromosomes , Cells, and Tissue Sections
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Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections

This chapter includes contributions from several leading scientists, from researchers in molecular biology to clinical pathologists, who practice in situ hybridization. Protocols are given for in situ hybridizations on widely varying substrates, e.g., chromosome spreads, chromosomes in suspension, single cells, paraffin-embedded tissue sections, ultrathin tissue sections, and whole mount preparations. H ybridization methods are described for both D N A and RN A targets. Applications covered include gene mapping, gene expression, developmental biology, tumor biology, cell sorting, clinical cytogenetics, and analysis of infectious diseases. These procedures use digoxigenin, biotin, and fluorochromes for labeling D N A, RN A and oligonucleotides. Labeling techniques include the classical methods, such as random primed D N A labeling, nick translation, and oligonucleotide tailing with terminal transferase, as well as more modern methods such as PC R and PRIN S, a novel and highly sensitive approach to in situ hybridization, in which the probe is labeled after its hybridization to the target nucleic acid. Please note that the protocols have been optimized by members of the individual laboratories and can be varied if necessary.
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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections
Contents
ISH to whole chromosomes
In situ hybridization to human metaphase chromosomes using DIG-, biotin- or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. 62 J. W iegant, D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands; U . M athieu and P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any; J. W ienberg and N . A rnold, I nstitute for A nthropology and H um an G enetics, U niv ersity of M unich, G erm any ; S. Popp and T. C rem er, I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any ; D . C . Ward, H um an G enetics D epartm ent, Yale U niv ersity, N ew H av en, C onnecticut, U SA ; S. Joos and P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any. Detection of chromosomal imbalances using DOP-PCR and comparative genomic hybridization (CGH). 72 S. Joos, B. Schtz , M . Bentz , and P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any. Identification of chromosomes in metaphase spreads and interphase nuclei with PRINS Oligonucleotide Primers and DIG- or rhodamine-labeled nucleotides. 79 R . Seibl, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any. Identification of chromosomes in metaphase spreads with DIG- or fluorescein-labeled human chromosome-specific satellite DNA probes. 88 G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any. Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension. 94 D . C eleda, U . Bettag, and C . C rem er, I nstitute for A pplied Physics and I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any. A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe. 97 E. R . Schm idt, I nstitute for G enetics, Johannes G utenberg-U niv ersity of M ainz , G erm any. Multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems. 100 E. J. M . Speel, F. C . S. R am eaek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands. DNA in situ hybridization with an alkaline phosphatase-based fluorescent detection system. 108 G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any.
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ISH to cells
Combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. 110 E. J. M . Speel, F. C . S. R am eaek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands.
In situ hybridization to mRNA in in vitro cultured cells with DNA probes. 116 D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands.
Identification of single bacterial cells using DIG-labeled oligonucleotides. 119 B. Z arda, R . A m ann, and K.-H . Schleifer, I nstitute for M icrobiology, Technical U niv ersity of M unich, G erm any.
ISH to tissues
Detection of HPV11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe. 122 J. R olighed and H . L indeberg, EN T-departm ent and I nstitute for Pathology A arhus U niv ersity H ospital, D enm ark . Detection of mRNA in tissue sections using DIG -labeled RNA and oligonucleotide probes. 126 P. Kom m inoth, D iv ision of C ell and M olecular Pathology, D epartm ent of Pathology, U niv ersity of Z rich, Sw itz erland. Detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. 136 H . Breitschopf and G . Suchanek , R esearch U nit for Experim ental N europathology, A ustrian A cadem y of Sciences, V ienna, A ustria. RNA-RNA in situ hybridization using DIG-labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. M . D e Block and D . D ebrouw er, Plant G enetic System s N .V., G ent, Belgium .
141
Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG-dUTP . 146 D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands.
In situ hybridization of DIG-labeled rRNA probes to mouse liver ultrathin sections. 148 D . Fischer, D . Weisenberger, and U . Scheer, I nstitute for Z oology I , U niv ersity of W rz burg, G erm any.
RNA in situ hybridization using DIG-labeled cRNA probes. 152 H . B. P. M . D ijk m an, S. M entz el, A . S. de Jong, and K. J. M . A ssm ann, D epartm ent of Pathology, N ijm egen U niv ersity H ospital, N ijm egen, T he N etherlands.
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W hole mount ISH

Localization of the expression of the segmentation gene hunchback in Drosophila embryos with DIG-labeled DNA probes. 158 D . Tautz , I nstitute for G enetics and M icrobiology, U niv ersity of M unich, G erm any. Detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probes. 162 N . Patel and C . G oodm an, C arnegie I nstitute of Washington, Em bryology D epartm ent, Baltim ore, M aryland, U SA . Whole mount fluorescence in situ hybridization (FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana. 165 Serge Bauw ens and Patrick Van O ostv eldt, L aboratory for G enetics and L aboratory for Biochem istry and M olecular C ytology, G ent U niv ersity, G ent, Belgium .
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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to w hole chrom osom es
In situ hybridization to human metaphase chromosomes using DIG -, biotin-, or fluorochromelabeled DNA probes and detection with fluorochrome conjugates
J. Wiegant, D epartm ent of C ytochem istry and C ytom etry, L eiden U niv ersity, N etherlands.
In recent years, a number of improvements in chromosomal in situ hybridization protocols have been achieved. These have allowed a fairly high success rate for single copy gene localization as well as competition in situ hybridization. H ere we present a detailed outline of the procedure that has been successfully applied in various laboratories. The procedures have been optimized for fluorescent detection. For the second edition of this manual, we have included new procedures and new illustrations for multicolor, multitarget fluorescent detection. The procedures have also been used with slight modifications by the groups of D r. P. Lichter, D r. J. Wienberg, D r. T. C remer and D r. D . Ward. The illustrative material they have provided below demonstrates the flexibility and wide applicability of the technique. At the end of the article, D r. Lichters group has provided a troubleshooting guide to help solve hybridization problems that may occur.
II. Denaturation and hybridization

For denaturation and hybridization three alternative protocols are given: A. For probes recognizing repetitive targets such as the alphoid sequences B. For probes recognizing unique sequences C. For probes or probe cocktails which contain repetitive sequences occurring throughout the genome (e.g., A lu sequences) [competition hybridization]
A. For repetitive probes
1 U sing digoxigenin- (D IG -), biotin-, or
I. Pretreatment of metaphase spreads on slides (optional)

1 Incubate metaphase spreads with 100 l
of 100 g/ ml RN ase A (in 2 x SSC ) under a coverslip for 1 h at 37C . 2 Wash slides 3 x 5 min with 2 x SSC . 3 D ehydrate slides in an ethanol series (increasing ethanol concentrations). 4 Incubate with 0.0050.02% pepsin in 10 mM H C l for 10 min at 37C . 5 Wash slides as follows: 2 x 5 min with PBS. O nce with PBS containing 50 mM MgC l2. 6 Post-fix for 10 min at room temperature with a solution of PBS containing 50 mM MgC l2 and 1% formaldehyde. 7 Wash with PBS and dehydrate (as in Step 3 above).
any fluorochrome-labeled nucleotide, label 1 g of D N A probe according to the procedures described in C hapter 4 of this manual. 2 Precipitate the labeled probe with ethanol. 3 Prepare a probe stock solution as follows: Resuspend the precipitated probe at a concentration of 10 ng/ l in a solution containing 60% deionized formamide, 2 x SSC , 50 mM sodium phosphate; pH 7.0. Incubate the tube for 15 min at 37 C with occasional vortexing until the precipitated D N A dissolves. 4 D ilute the probe from the stock to the desired concentration (typically 2 ng/ l) in a solution containing 60% deionized formamide, 2 x SSC , 50 mM sodium phosphate; pH 7.0. 5 Apply 5 l diluted probe under a cover slip on the object slide and place the slide in an oven at 80C for 24 min to denature the probe and target. N ote: Sealing w ith rubber cem ent is not m andatory. 6 To hybridize, place the slides in a moist chamber at 37 C overnight. 7 Wash the slide as follows: 3 x 5 min at 37C with 2 x SSC containing 60% formamide. 1 x 5 min with the buffer to be used for immunological detection.
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B. For unique probes

1 Prepare the probe stock solution as
3 Prepare a probe stock solution by
described under Procedure IIA, but use a solution containing 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. 2 D ilute 25 l of the probe stock solu tion to 10 l with a solution containing 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. Mix well! 3 To denature probe and target, do the following: Apply 10 l of the dilute hybridization solution under a coverslip on the object slide. Seal coverslip to slide with rubber cement. Place the slide in an oven at 80C for 24 min. 4 To hybridize, place the slides in a moist chamber at 37 C overnight. 5 Wash the slide as follows: 3 x 5 min at 45C with 2 x SSC containing 50% formamide. 5 x 2 min with 2 x SSC . 1 x 5 min with the buffer used in detection.
C. For probes containing repetitive elements [competitive hybridization]
1 U sing D IG -, biotin-, or any fluoro-
chrome-labeled nucleotides, label 1 g of D N A probe according to the procedures described in C hapter 4 of this manual. 2 To precipitate the labeled probe, do the following: Add a 50-fold excess of human C o t-1 D N A (or 500-fold excess fragmented human placenta D N A), a 50-fold excess of fragmented herring sperm D N A, a 50-fold excess of yeast tRN A, 1/ 10th volume of 3 M sodium acetate ( pH 5.6), and 2.5 volumes of cold (20C ) ethanol. Incubate for 30 min on ice. C entrifuge for 30 min at 4C . D iscard the supernatant and dry the pellet.
dissolving the pellet in a solution of 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. D epending on the probe cocktail, the final concentration ranges from 1040 ng/ l. Mix well! Examples: Prepare a 10 ng/ l stock solution of cosm id probes, a 20 ng/ l stock of chrom osom e specific libraries, or a 40 ng/ l stock of Y A C probes. 4 For hybridization, dilute the probe from the stock to the desired concentration in a solution of 50% deionized formamide, 2 x SSC , 10% dextran sulfate, 50 mM sodium phosphate; pH 7. Examples: For hybridiz ation, use 25 ng/ l of cosm id probes, 1020 ng/ l of chrom osom e specific libraries, or 2040 ng/ l of Y A C probes. 5 D enature the probe at 75C for 5 min, chill on ice, and allow annealing of the repetitive elements at 37C for either 30 min (if C o t-1 D N A is used as competitor) or 2 h (if total human placenta D N A is used as competitor). 6 Prepare the chromosomes on the slide by performing the following steps: To the chromosomes, add a solution of 70% formamide, 2 x SSC , 50 mM sodium phosphate; pH 7. C over with a coverslip. Incubate in an oven at 80 C for 24 min to denature the chromosomes. Remove the coverslip and quench the chromosomes in chilled 70% ethanol. Dehydrate the slide by passing it through 90% ethanol, then 100% alcohol. Air dry the slide, preferably on a metal plate at 37 C . 7 H ybridize the chromosomes overnight with 10 l of the pre-annealed probe (from Step 5) under a sealed coverslip. 8 Wash the slide as follows: 3 x 5 min at 45C with 2 x SSC containing 50% formamide. 3 x 5 min at 60C with 0.1 x SSC . 1 x 5 min with the buffer to be used in immunological detection.
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III. Single color fluorescent detection with immunological amplification

A1. Biotin-labeled probe, low sensitivity
1 Wash slides briefly with TN T buffer
B1. Digoxigenin-labeled probe, low sensitivity

[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween 20]. 2 Incubate for 30 min at 37C with TN B buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. 3 Incubate for 30 min at 37C with the proper dilution (typically 10 g/ ml) of a fluorescently labeled streptavidin conjugate in TN B. 4 Wash the slides (3 x 5 min) with TN T. 5 Prepare the slides for viewing by per forming the following steps: D ehydrate through an ethanol series (70% , then 90% , then 100% ethanol; 5 min each). Air dry. Stain with the appropriate D N A counterstain. Embed in Vectashield (Vector). 6 View the slides by fluorescence micro scopy, using appropriate filters.
A2. Biotin-labeled probe, high sensitivity
[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween 20]. 2 Incubate for 30 min at 37C with TN B buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. 3 Incubate for 30 min at 37C with the proper dilution (typically 2 g/ ml, in TN B) of a fluorescently labeled sheep anti-D IG antibody. 4 Wash the slides (3 x 5 min) with TN T. 5 Prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.
B2. Digoxigenin-labeled probe, high sensitivity
[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween 20]. 2 Incubate for 30 min at 37C with TN B buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. 3 Incubate for 30 min at 37C with the proper dilution (typically 10 g/ ml) of a fluorescently labeled streptavidin conjugate in TN B. 4 Wash the slides (3 x 5 min) with TN T. 5 Incubate the slides for 30 min at 37C with the proper dilution of biotinylated goat anti-streptavidin (5 g/ ml) in TN B. 6 Repeat the washes as in Step 4. 7 Repeat Step 3. 8 Repeat the washes as in Step 4. 9 Prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.
[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween 20]. 2 Pipette 100 l of TN B buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent] onto each slide and cover with a 24 x 50 mm coverslip. Incubate for 30 min at 37C in a moist chamber (1 L beaker containing moistened tissues, covered with aluminum foil). 3 Immerse the slides for 5 min in TN T to loosen the coverslips. 4 Prepare fresh working solutions of three antibodies: Antibody 1 working solution: mouse monoclonal anti-D IG , 0.5 g/ ml in TN B. Antibody 2 working solution: D IG conjugated sheep anti-mouse Ig, 2 g/ ml in TN B. Antibody 3 working solution: fluorescein- or rhodamine-conjugated sheep anti-D IG , 2 g/ ml in TN B. N ote: T he three antibodies used in this procedure are also av ailable in the Fluorescent A ntibody Enhancer Set for D I G D etection. 5 Pipette 100 l of antibody 1 working solu tion onto each slide and cover with a 24 x50mm coverslip.Incubatefor 30min at 37C in a moist chamber.
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6 Wash the slides (3 x 5 min) at room
7 Pipette 100 l of antibody 2 working
temperature with TN T.
IV . Multicolor fluorescence in situ hybridization (Multicolor FISH)

For multicolor FISH , several sets of probes with different labels must be hybridized simultaneously to the target and visualized with combinations of antibodies. Table 1 lists an antibody-probe matrix which allows a triple color detection of three differently labeled chromosome specific libraries (so-called chromosome painting probes). Incubation 1 2 3 Probe 1 Biotin StreptavidinTexas Red Biotin-labeled anti-streptavidin StreptavidinTexas Red Probe 2 D igoxigenin Mouse antiD IG antibody Probe 3 Fluorescein Rabbit antifluorescein antibody
solution onto each slide and add a 24 x 50 mm coverslip. Incubate for 30 min at 37 C in a moist chamber. 8 Repeat the washes as in Step 6. 9 Pipette 100 l of antibody 3 working solution onto each slide and add a 24 x 50 mm coverslip. Incubate for 30 min at 37 C in a moist chamber. N ote: For ev en higher sensitiv ity, repeat Steps 58 before perform ing the incubation w ith antibody 3 (Step 9). 10 Repeat the washes as in Step 6. 11 Prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.
C1. Fluorescently labeled probes (fluorescein, coumarin, CY3, rhodamine, Texas Red), low sensitivity
Sheep anti-mouse FITC -labeled goat antibody anti-rabbit antibody AMC A-labeled sheep anti-D IG antibody
After the posthybridization washes (Procedure II), prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.
C2. Fluorescein-labeled probes, high sensitivity
N ote: Be careful that the selected antibodies do not cross-react.

Outline of protocol for multicolor FISH
1 Simultaneously hybridize all probes
2 For each of the 3 detection incubations,
[100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.05% Tween 20]. 2 Incubate for 30 min at 37C with TN B buffer [100 mM Tris-H C l (pH 7.5), 150 mM N aC l, 0.5% blocking reagent]. 3 Incubate for 30 min at 37C with the proper dilution of an anti-fluorescein antibody (either polyclonal or monoclonal) in TN B. 4 Wash the slides (3 x 5 min) with TN T. 5 Depending on the antibody used in Step 3, incubate with the proper dilution of a fluorescently labeled rabbit anti-mouse or goat anti-rabbit antibody in TN B for 30 min at 37 C . 6 Repeat the washes as in Step 4. 7 Prepare the slides for viewing as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.
listed in Table 1 to the target.
perform the following steps: Prepare the antibodies needed in the incubation (as listed in Table 1) at the proper dilution in the correct buffer. N ote: U se the procedures in Section I I I abov e as a guideline. Mix all antibodies needed in the incubation and incubate simultaneously with the slide. Perform blocking and washing steps as in Section III above. 3 Repeat Step 2 until all incubations are complete. 4 D o not counterstain the slides, but dehydrate, air dry, and mount the slides as in the biotin low sensitivity procedure (Procedure IIIA1), Step 5.
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V . Results obtained with human metaphase chromosome spreads

The following examples (from our laboratory and other laboratories) show different applications of the procedures described in this section .
A. Multicolor FISH of human lymphocyte metaphase and interphase cells from the D epartm ent of C ytochem istry and C ytom etry, L eiden U niv ersity, N etherlands
B. Detection of a trisomy in a leukemia patient in interphase nuclei using double in situ hybridization from U . M athieu and D r. P. L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any.
The techniques described above allow the detection of two, three, or many different probes simultaneously (Figures 14).
Figure 1: FISH of CY3-labeled satelliteIII probepUC1.77(chromosome 1) to human lymphocyte metaphase and interphase cells. The slides were counterstained with YO YO-1. The photomicrograph was taken with a double band-pass fluorescence filter to allow simultaneous visualization of fluorescein and Texas Red.
N ick translation was used to label a chromosome 17-specific alphoid DN A (with digoxigenin) and a chromosome 18-specific alphoid DN A (with biotin). Both the digoxigenin-labeled and the biotin-labeled probes were hybridized to a chromosome sample from a leukemia patient. The procedures described above for repetitive DN A (Procedure IIA of this article) were used for slide preparation, labeling, and hybridization, except the following minor modifications were necessary for alphoid DN A:
1 After nick translation labeling, concen-
trate the probe (approx. 1030 ng D N A) by drying the D N A directly in the tube. N ote: Ethanol precipitation is not necessary.
2 D issolve the dried D N A pellet in 60%
Figure 2: Triple color FISH of coumarin-labeled satellite III probe pUC1.77 (chromosome 1), fluorescein-labeled alphoid probe pBamX5 (chromosome X), and rhodamine-labeled alphoid probe p17H8 (chromosome 17) to human lymphocyte metaphase and interphase cells. The photomicrograph was taken by superimposing the coumarin, fluorescein, and rhodamine images.
formamide, 2 x SSC . N ote: D o not use dextran sulfate in the resuspension buffer. O m ission of dextran sulfate reduces the chance of cross-hybridiz ation.
3 After hybridization, use the following
washes: 3 x 5 min at 42C with 60% formamide, 2 x SSC ; pH 7. N ote: C om pared to the standard protocol the concentration of form am ide is increased up to 60% .
Figure 3: Double color FISH of a biotin-labeled chromosome1-specificlibraryanda digoxigeninlabeled chromosome 4-specific library to human lymphocyte metaphase and interphase cells. The libraries were visualized by a combined immunocytochemical reaction using fluoresceinlabeled anti-digoxigenin and Texas Red-labeled streptavidin. The photomicrograph was taken with a double band-pass fluorescence filter to allow simultaneous visualization of fluorescein and Texas
Figure 4: Twelve color FISH of 12 different ratiolabeled chromosome-specific libraries to human lymphocyte metaphase and interphase cells. For details of the experiment, see Dauwerse et al. (1992). The photomicrograph was created by superimposing the coumarin image and the image obtained with a double band-pass fluorescence filter (to allow simultaneous visualization of fluorescein and Texas Red).
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Figure 5 shows the result of the hybridization. The digoxigenin-labeled probe was visualized with anti-DIG-rhodamine; the biotin-labeled probe, with avidin-FITC.
D. Interphase nuclei of a patient with Ph1-positive ALL(acute lymphoblastic leukemia) from M . Bentz , P. L ichter, H . D hner and G . C abot.
The Philadelphia chromosome (Ph1) is the derivative of a translocation between chromosome 9 and chromosome 22 [(t9;22) (q34;q11)]. Figure 7 shows the use of two probes, one specific for chromosome 9 and the other specific for chromosome 22, to detect Ph1.
Figure 7: Double color FISH to
Figure 5: Double color FISH of a digoxigeninlabeled chromosome 17-specific probe and a biotin-labeled chromosome 18-specific probe to a human chromosome preparation containing a trisomy. Each alphoid DNA probe was specific for the centromere of its target chromosome. In the metaphase and in the interphase, the probes detected three signals (green) from chromosome 18 and two signals (red) from chromosome 17. DAPI was used for counterstaining. detect the Philadelphia chromosome (Ph1) in a patient with ALL. A cosmid probe specific for chromosome 9 and a Y AC probe specific for chromosome 22 were hybridized to interphase nuclei (Bentz et al., 1994). These two probes detected two red (rhodamine) signals from chromosomes 22, one green (FITC) signal from chromosome 9 and one yellow signal caused by the overlapping of red (chromosome 22) and green (chromosome 9) signals on Ph1. DAPI was used for counterstaining.
Reprinted from Bentz et al. (1994) Detection of Chimeric BCRABL genes on Bone Marrow Samples and Blood Smears in Chronic Myeloid and Acute Lymphoblastic Leukemia by In Situ Hybridization, Blood 83 (7); 19221928 with permission of the Journal Permissions Department, W . B. Saunders Company.
C. Detection of a trisomy and a tetrasomy in interphase nuclei of a leukemia patient with two different cosmid probes from U . M athieu and D r. P. L ichter
A digoxigenin-labeled cosmid probe specific for chromosome 14 and a biotin-labeled cosmid probe specific for chromosome 21 were used to detect polyploidy in a leukemia patient (Figure 6). Probes were labeled by nick translation and visualized with either anti-D IG -rhodamine (chromosome 14-specific probe) or avidin-FITC (chromosome 21-specific probe).
E. Chromosomal in situ suppression (CISS) hybridization of the human DNA library specific for chromosome 2 to chromosomes of Gorilla gorilla from D r. J. Wienberg and D r. N . A rnold, I nstitute for A nthropology and H um an G enetics, U niv ersity of M unich, G erm any.
The hybridization pattern (Figure 8) obtained with specific D N A probe sets (Weinberg et al., 1990) confirms the assumed fusion of two submetacentric chromosomes during human evolution.
Figure 6: Double color FISH of a digoxigeninlabeled chromosome 14-specific cosmid probe and a biotin-labeled chromosome 21-specific cosmid probe to a human chromosome preparation of a leukemia patient containing a trisomy and a tetrasomy. In the interphase nuclei, the cosmid DNA probes detected three copies (red) of chromosome 14 and four copies (green) of chromosome 21. DAPI was used for
Figure 8: FISHof a chromosome 2-specific library to chromosomes of Gorilla gorilla. Standard chromosome preparations wereobtainedfromEBVtransformed lymphoblastoid cell lines. DNAprobes were from flow-sorted human chromosomes cloned to phages or plasmids. Probes were labeled with digoxigenin by nick translation. Detection was performed with monoclonal mouse anti-DIGantibody, rabbit anti-mouse-TRITC, and goat anti-rabbit-TRITC.
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F . Illustration of a chromosomal translocation from a patient with chronic myeloid leukemia from S. Popp and D r. T. C rem er, I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any.
The chromosomal translocation was detected by using library probes from sorted chromosomes 9 and 22 (Figure 9). In Panel a, the chromosome 22-specific probe has
clearly painted the normal chromosome 22 (bottom), the Philadelphia chromosome (triangle) and the translocated material 22q11 ! 22qter contained in the derivative chromosome 9 (arrow). In Panel b, the chromosome 9-specific probe has stained the normal chromosome 9 blue except for the centromeric region, while in the derivative chromosome 9, the region 9pter ! 9q34 is also painted. The arrow points to the breakpoint on chromosome 9.
"
"
5
Figure 10: FISH of an ATPase specific probe and a probe containing L1-repetitive sequences to female mouse chromosomes. Y ellow color (banding) is due to the biotin-labeled probe containing L1-repetitive sequences, which was detected with FITC. Red dots indicate a signal from each of the four (look closely) chromatids obtained with the digoxigenin-labeled ATPasespecific probe, which was detected with anti-DIG Fab fragments (from sheep) and Texas Red-conjugated anti-sheep Ig Fab. The blue color is the DAPI counterstain.
Figure 9: Two-color chromosomal in situ suppression (CISS)-hybridization of a metaphase spread obtained from a patient with chronic myeloid leukemia 46, XY , t(9;22) (q34;q11). In Panel a, the probe was a digoxigenin-labeled chromosome 22-specific library, which was detected with a mouse anti-DIG antibody and a FITC-conjugated sheep anti-mouse antibody. The same metaphase spread was simultaneously painted (Panel b) with a biotin-labeled chromosome 9-specific library, which was detected with avidin conjugated to the fluorochrome AMCA (aminocoumarin). Chromosomes were counterstained with propidium iodide. Chromosomal analysis using G TG -banded chromosomes was kindly performed by R. Becher, W estdeutsches Tumorzentrum, Essen.
"
G. Digitized images of female mouse chromosomesvisualizedwitha Zeiss epifluorescent microscope equipped with a cooled CCD camera from D r. D . C . Ward, H um an G enetics D epartm ent, Yale U niv ersity, N ew H av en, C T, U SA .
68
"
In Figure 10, a probe specific for N a+/ K + ATPase alpha subunits and a probe containing an L1-repetitive sequence were hybridized to female mouse chromosomes.
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Reagents available from Boehringer Mannheim for these procedures

Reagent DIG-Nick Translation Mix* for in situ probes Description 5x conc. stabilized reaction buffer in 50% glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mMdATP , 0.25 mMdCTP , 0.25 mMdG TP , 0.17 mM dTTP and 0.08 mM DIG -11-dUTP . 5x conc. stabilized reaction buffer in 50% glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mM dATP , 0.25 mM dCTP , 0.25 mM dG TP , 0.17 mM dTTP and 0.08 mM biotin-16-dUTP . 5x conc. stabilized reaction buffer in 50% glycerol, DNA Polymerase I and DNase I. Set of dATP , dCTP , dG TP , dTTP , 100 mM solutions, lithium salts. Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Pyrimidine specific endoribonuclease which acts on single-stranded RNA. Aspartic endopeptidase with broad specificity. COT-1 DNA is used in chromosomal in situ suppression [CISS] hybridization. The ready-to-use solution is directly added to the hybridization mix. Lyophilizate, 1 mg of dry substance corresponds to 16 A260 units. Aqueous solution, 10% (w/v) Powder Cat. No. 1745 816 Pack size 160 l (40 labeling reactions) 160 l (40 labeling reactions) 200 l 4x10 mol (100 l) 25 nmol (25 l) 25 nmol (25 l) 25 nmol (25 l) 25 mg 100 mg 1g 5g 500 g (500 l) 500 mg (50 ml) 100 mg 500 mg 5x10 ml 100 g 500 g 1 kg 50 g
Biotin-Nick Translation Mix* for in situ probes
1745 824
Nick Translation Mix* for in situ probes dNTP Set Fluorescein-12-dUTP Tetramethylrhodamine-6-dUTP AMCA-6-dUTP RNase A Pepsin DNA, CO T-1, human DNA, MB-grade from fish sperm tRNA from bakers yeast Tween 20 Tris
1745 808 1 277 049 1 373 242 1 534 378 1 534 386 109 142 109 169 108 057 1 693 387 1 581 074 1 467 140 109 495 109 509 1 332 465 127 434 708 968 708 976 1 096 176
Blocking Reagent, for nucleic acid hybridization Anti-Digoxigenin, clone 1.71.256, mouse IgG 1, Anti-DigoxigeninFluorescein, Fab fragments from sheep Anti-DigoxigeninRhodamine, Fab fragments from sheep Anti-Mouse Ig-Digoxigenin, F(ab)2 fragment Streptavidin-AMCA
Powder
For the detection of digoxigenin-labeled compounds For the detection of digoxigenin-labeled compounds For the detection of digoxigenin-labeled compounds Antibody for triple color FISH For the detection of biotin-labeled compounds.
1 333 062
100 g
1 207 741
200 g
1 207 750
200 g
1 214 624 1 428 578 1 055 097 236 276
200 g 1 mg 1 mg 10 mg
Streptavidin-Fluorescein For the detection of biotin-labeled compounds. DAPI Fluorescence dye for staining of 4',6-Diamidine-2'-Phenyl- chromosomes indole Dihydrochloride
*This product or the use of this product may be covered by one or more patents of Boehringer Mannheim G mbH, including the following: EP patent 0 649 909 (application pending).
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VI. T roubleshooting guide for in situ hybridization on chromosome spreads

from Stefan Joos and Peter L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any. U se the following tips to diagnose and correct commonly occurring problems in the FISH protocols described above.
A. If no signal is observed, then:
1 Amplify signal. 2 C heck probe labeling by performing the
I f the probe is larger than 500 nt,
add more D N ase I (roughly about 110 ng) to the probe sample kept on ice, then incubate longer at 15C . N ote: U sually higher concentrations of D N ase m ust be added for an additional 30 m in incubation. A fter the incubation, analyz e the probe on a gel as abov e.
I f the D N A is almost or completely
following dot blot assay: Spot serial dilutions of labeled control. Spot serial dilutions of labeled sample. C ompare intensities of the spots. N ote: For the detailed procedure on how to estim ate the labeling efficiency please refer to C hapter 4, I X , page 51.
3 After labeling the probe according to
Steps 14 of the nick translation procedure (Procedure III) in C hapter 4 of this manual, check the size of the labeled probe molecules as follows: To a 510 l aliquot of probe, add gel loading buffer and denature the probe by incubating it in a boiling water bath for 3 min, then cool it on ice for 3 min. N ote: Keep the rem ainder of the probe sam ple on ice w hile running the gel.
Load the aliquot on a standard 12%
agarose minigel along with a suitable size marker. Q uickly run the gel (e.g. 15 volts per cm for 30 min) to avoid renaturation of the probe in the gel. Visualize D N A in the gel, e.g. by staining gel in 0.5 g/ ml ethidium bromide, and take photograph during U V illumination. N ote: T he probe m olecules w ill be v isible as a sm ear. T he sm ear should contain only fragm ents sm aller than 500 nucleotides (nt) and larger than 100 nt. A peak intensity at 250 300 nt seem s optim al.
D epending on the size of the probe
undigested, purify the probe and repeat the labeling step. I f part of the D N A is smaller than 100 nt, repeat the labeling step using less D N ase I. I f all the D N A is smaller than 100 nt, repurify the probe (to remove any possible contaminating D N ase) and repeat the labeling step. 4 C heck whether the fluorochromes have separated from the detecting molecules by passing the solution of fluorochromeconjugated molecules through a Q uick Spin column (G -50). Then determine the state of the molecules: I f color remains in flow through, fluorochromes are still conjugated. If color remains in the upper part of the column, fluorochromes have separated from the detecting molecules. 5 Modify chromosome denaturation procedure (Procedure II in this article) by either varying the time and temperature of the denaturation step or by further pepsin digestion according to the following protocol: N ote: D igestion w ith pepsin can significantly im prov e probe penetration, but ov erdigestion can also occur. Pepsin treatm ent is often useful w hen specim en preparations are suboptim al, e.g., in m any clinical sam ples.
Mark the area of interest on the back Wash slide in 2 x SSC at 37 C for Apply 120 l RN ase A solution
of the slide using a diamond stylus. 5 min (in a C oplin jar).
molecules, do one of the following: I f the D N A is between 100500 nt, proceed with the ED TA inactivation of the reaction mixture (as in Step 7 of the nick translation procedure, C hapter 4).
(100 g/ ml RN ase A in 2 x SSC ) to slide, cover with larger coverslip (e.g. 22 x 50 mm), and incubate for 1 h at 37C in a moist chamber. Wash slide in 2 x SSC (or PBS) for 3 x 5 min (C oplin jar). Wash slide in prewarmed PBS for 5 min at 37 C .
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Incubate slide in pepsin solution
(10 mg pepsin in 100 ml 10 mM H C l) for 10 min at 37 C (in a water bath). Wash slide in PBS for 5 min at room temperature (RT). Incubate slide for 10 min at RT in a solution of 1% acid-free formaldehyde, PBS, 50 mM MgC l2. Wash slide again in PBS for 5 min at RT. D ehydrate slide in a series of ethanol baths (70% , then 90% , then 100% ; ! 5 min each) and air dry. 6 C heck quality of the formamide. D eionize formamide (several batches from several sources should be tested) using ion exchange resin (e.g. D onex XG 8). 7 C heck microscope. Be sure that mercury (H g2) lamp of the microscope is in good condition and well adjusted. O ld lamps dont have good excitation properties. 8 Perform control experiments with alphoid D N A probes.
B. If the hybridization signal fades, then:
1 C hange to a different batch of fluoro-
D. If there is a starry background fluorescence, then:
Possible cause Agglutination of fluorochromes coupled to antibodies
Remedy Perform a control experiment in the absence of probe to see if you get a non-specific signal from the fluorochrome-conjugated antibody alone. I f you do, spin the detection solution briefly and take only the supernatant. Alternatively pass the detection solution through a G -50 Q uick Spin column to remove the agglutinates. C heck the size of the probe as described in Step A3 above and label again.
Labeled probe molecules are too long
E. If there is a general strong staining of chromosomes and nuclei, then:

C heck the size of the labeled probe (as in
Step A3 above). It is most likely too small. I f the probe is too small, repurify (to remove any contaminating D N ase) and relabel the probe.
References
Bentz, M.; Cabot, G .; Moos, M.; Speicher, M. R., G anser, A.; Lichter, P .; Dhner, H. (1994) Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood 83, 19221928. Dauwerse, J. G .; Wiegant, J.; Raap, A. K.; Breuning, M. H.; Van Ommen, G . J. B. (1992) Multiple colors by fluorescence in situ hybridization using ratiolabeled DNA probes create a molecular karyotype. Hum. Mol. G enet. 1, 593598. Wienberg, J.; Jauch, A.; Stanyon, R.; Cremer, T . (1990) Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. G enomics 8, 347350.
chrome-conjugated antibody. with a secondary and/ or tertiary antibody to increase the signal. N ote: Be aw are of the back ground, w hich w ill also be am plified. Before amplification, remove embedding medium by washing (2 x SSC, 3742C, 4 x 10 min). 3 Try different antifading solutions [e.g. 1,4-diazobicyclo-(2,2,2)-octane (DAPCO ) or Vectastain (Vector)].
2 Amplify
C. If there is a milky background fluorescence, then:
Possible cause Inadequate quality of chromosome preparation Insufficient blocking D irty glass slides Bad quality of embedding medium
Remedy Treat with pepsin to remove cell debris (see protocol in Step A5 above). Try to block with different reagents such as 3% BSA, human serum, or 35% dry milk. Wash the slide with ethanol and rinse with water before spreading chromosomes. C hange embedding medium.
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Detection of chromosomal imbalances using DO PPCR and comparative genomic hybridization (CG H)
Stefan Joos, Barbara Schtz , M artin Bentz , and Peter L ichter, G erm an C ancer R esearch C enter, H eidelberg, G erm any.
Recently, a new approach of fluorescence in situ hybridization was introduced, called comparative genomic hybridization (CGH ) (Kallioniemi et al., 1992), which allows the comprehensive analysis of chromosomal imbalances in entire genomes (Du Manoir et al., 1993; Joos et al., 1993; Kallioniemi et al., 1992; Kallioniemi et al., 1993). The principle of CGH is shown in Figure 1. The genomic DN A from cell populations to be tested, such as fresh or paraffin-embedded tumor tissue, is labeled with modified nucleotides (e.g., biotinylated dUTP) and used as a probe for in situ hybridization to normal metaphase chromosomes. This probe is called test DN A. As an
total genomic DNA from cells to analyze (test DNA) labeled with biotin total genomic DNA from normal cells (control DNA) labeled with digoxigenin
By comparing the hybridization patterns of test and control DN A, the changes in signal intensities caused by imbalances within the analyzed tissue can be identified. Thus, C G H provides a comprehensive picture of all chromosomal gains and losses within a single experiment. In contrast to banding analysis, C G H requires no preparation of metaphase chromosomes from the cells to be analyzed (which in certain tumor types may be difficult or even impossible). In many cases, only small amounts of tumor tissue are available for cytogenetic analysis. Therefore it is advantageous to combine C G H with universal PC R protocols (as shown in Figures 4 and 5 under Results) (Speicher et al., 1993). Both sequence independent amplification (SIA) (Bohlander et al., 1992) and D O P-PC R (degenerate oligonucleotide primer PC R) (Telenius et al., 1992) can be combined with C G H . As is shown in Figure 2, the D O P primer contains a central cassette of 6 degenerated nucleotides and is flanked by specific sequences at the 3' end (6 nucleotides) and at the 5' end (10 nucleotides). The amplification procedure is divided into two steps. D uring the first step, five PC R cycles are performed at low stringency conditions (annealing temperature, T a = 30C ). These conditions allow a frequent annealing of the D O P primer, which is primarily mediated by the short specific sequence at its 3' end and by the adjacent central cassette of degenerated nucleotides. Eventually a pool of many different D N A sequences is generated which should represent a statistically distributed subpopulation of the genomic D N A. D uring the second step (35 cycles) the annealing temperature is raised to 62C , allowing amplification only after specific base pairing of the full length primer sequence. Therefore, all the sequences that have been synthesized in the first step are now exponentially amplified, resulting in a large amount of D N A that can be used as probe for C G H .
co-hybridization (normal chromosomes)
detection DNA from: cells with amplified DNA or polysomies cells with deletions or monosomies results in signals on normal chromosomes (partial karyotype) signals from hybridized test DNA signals from co-hybridized control DNA
Figure 1: Schematic illustration of comparative genomic hybridization (CGH) for chromosome analysis. For explanation, see text.
internal control, genomic DN A derived from cells with a normal karyotype is differentially labeled and hybridized simultaneously (control DN A). For detection of the hybridized test and control DN A, different fluorochromes are used, and each is visualized by epifluorescence microscopy with selective filters. H ybridization with genomic DN A results in a general staining of all chromosomes. H owever, if the tissue analyzed harbors additional chromosomal material (e.g., a trisomy, amplifications etc.), hybridization reveals higher signal intensities at the corresponding target regions of the hybridized chromosomes (Figure 1). Correspondingly, deletions in the test sample are visible as lower signal intensities (Figure 1).
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Protocols for DO P-PCR and CGH analysis are described below. Amplified sequences can be detected directly by visual inspection using an epifluorescence microscope (Results Figures 3 and 4). H owever, the comprehensive analysis of gains and losses of single chromosomes (or parts thereof) requires digital image analysis with sophisticated software programs. For further description of the evaluation of CGH experiments as well as the different steps of the CGH protocol, see the literature (Du Manoir et al., 1995; Kallioniemi et al., 1994; Lichter et al., 1994; Lundsteen et al., 1995; Piper et al., 1995).
DOP-PCR Telenius et al., 1992 5 CCG ACTCG AGNNNNNN ATG TG G 3
1.
Low stringency PCR (T a = 30 C; 5 cycles) ! frequent priming at multiple sites 5 3
I. Preparation and labeling of DO P-PCR products

1 Prepare a D N A template by isolating
genomic D N A from small amounts of tissue (Maniatis, Fritsch, and Sambrook, 1986). O ne mg of tissue results in about 1 g of genomic D N A. N ote: See the literature (Kaw asak i, 1990; Speicher et al., 1993) for protocols on D N A isolation from sm aller am ounts of tissue (dow n to only a few hundred cells) or from paraffin-em bedded m aterial. 2 For each reaction, mix the following components in a PC R reaction tube on ice: 5.0 l 10 x concentrated D O P-PC R buffer (20 mM MgC l2; 500 mM KC l; 100 mM Tris-H C l, pH 8.4; 1 mg/ ml gelatin). 5.0 l 10 x concentrated nucleotide mix (dATP, dC TP, dG TP, dTTP, 2 mM each). 110 l genomic D N A (0.11 ng/ l). 5.0 l 10 x concentrated D O P-PC R primer [5'-C C G AC TC G AG N N N N N N ATG TG G -3' (N =A,C ,G , or T), 20 M]. enough sterile water to make a final reaction volume of 50 l. 0.25 lTaq D N A Polymerase (5 units/ l) (should be added last). 3 Prepare a negative control by combining the same solutions as in Step 2, but omitting the template D N A. 4 O verlay reaction mixtures with 50 l mineral oil.
2.
Higher stringency PCR (T a = 62 C; 35 cycles) ! specific priming of pre-amplified sequences
Figure 2: Schematic illustration of DOP-PCR. For explanation, see text.

5 Transfer the reaction tubes to a thermo
cycler and start the PC R. U se the following thermal profile: Initial denaturation: 10 min at 94C . 5 C ycles, each consisting of: 1 min denaturation at 94C 1.5 min annealing (low stringency) at 30 C 3 min temperature ramping, from 30 C to 72C 3 min elongation at 72C . 35 C ycles, each consisting of: 1 min denaturation at 94C 1 min annealing (high stringency) at 62 C 3 min elongation at 72 C , with the elongation time increased by 1 second every cycle [i.e., the 1st high stringency cycle has an elongation tim e of 3 m in; the 2nd, 3 m in and 1 s; the 3rd, 3 m in and 2 s, etc.] Final elongation: 10 min at 72C .
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6 Analyze aliquots of the reaction mix
4 C o-precipitate the probe D N As in each
tures on an agarose gel by performing the following steps: Mix the aliquots with gel loading buffer (0.25% bromophenol blue and 30% glycerol). Load samples on a minigel. As a size marker use a 1 kb ladder or other suitable size standard. Run the gel in 1x TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM ED TA; pH 8.0) for 30 min at 100 volts. 7 Stain the gel with ethidium bromide and photograph. In the reaction samples, you should see a smear of D N A, ranging in size from about 200 bp to 2000 bp. N o smear should be visible in the negative control. 8 C ollect the amplified D N A products from the reaction mix by using the H igh Pure PC R Product Purification Kit, see page 50. 9 Label the amplified D N A by standard nick translation procedures (Lichter and C remer; Lichter et al., 1994).
II. Comparative genomic hybridization (CG H)

1 Prepare slides of normal metaphase
chromosomes as described in the literature (Lichter and C remer; Lichter et al., 1990). 2 Prepare the following solutions: 3 M sodium acetate, pH 5.2. D eionized formamide (molecular biology grade): For deionization, use ion exchange resin. The conductivity of the formamide should be below 100 Siemens. H ybridization buffer (4 x SSC , 20% dextran sulfate): Prepare 20 x SSC . C arefully dissolve dextran sulfate in water to make a 50% dextran sulfate solution. Autoclave the dextran sulfate solution or filter it through a nitrocellulose filter. Mix 200 l of 20 x SSC , 400 l of 50% dextran sulfate and 400 l of double-distilled water. Store the hybridization buffer at 4C until you use it. 3 To prepare probe D N A, combine 1 g of each labeled test and control D N A with 50100 g of human C o t-1 D N A.
tube by adding 1/ 20 volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol. Mix well and incubate at 70C for 30 min. 5 Spin the precipitated D N A in a microcentrifuge at 12,000 rpm for 10 min at 4C . D iscard the supernatant and wash the pellet with 500 l of 70% ethanol. 6 Spin the precipitated D N A again (12,000 rpm, 10 min, 4C ). D iscard the supernatant and lyophilize the pellet. 7 Add 6 l deionized formamide to the lyophilizate and resuspend the probe D N A by vigorously shaking the tube for > 30 min at room temperature. 8 Add 6 l of hybridization buffer to the tube containing probe D N A and again shake for > 30 min. 9 D enature and dehydrate normal metaphase chromosomal D N A on slides as described in the literature (Lichter and C remer; Lichter et al., 1990). 10 Prepare the probe (from Step 8) for hybridization by performing the following steps: D enature probe D N A at 75C for 5 min. Preanneal repetitive sequences by incubating the probe at 37C for 2030 min. 11 Add prepared probe (from Step 10) to denatured chromosomal spreads (from Step 9). Proceed with in situ hybridization and probe detection as described in the literature (Lichter and C remer; Lichter et al., 1990). 12 Evaluate the C G H experiment with an epifluorescence microscope.
Results
The C G H procedure was used to reveal chromosomal imbalances (Figure 3) in a T-cell prolymphocytic leukemia (T-PLL). C hromosomal regions 6q, 8p, and 11qter are stained more weakly in the tumor D N A than in the control D N A, indicating deletions of all or part of the chromosomal arms in the tumor D N A. C G H also identified overrepresented chromosomal regions (6p, 8q, and 14q) which stain more heavily in the tumor than in the control.
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Figure 3: Comparative genomic
hybridization (CGH) experiment revealing chromosomal imbalances in a T-cell prolymphocytic leukemia (T-PLL). DNA from tumor cells was labeled with biotin and detected via a FITClabeled antibody (green) whereas the control DNA was labeledwith digoxigenin and detected via a rhodamine-labeled antibody (red). For explanation, see the text. This photograph was taken by S. Joos and P . Lichter with permission of Springer Verlag, Heidelberg and New York.
Figure 4: Detection of amplified
sequences in the genomic DNA of patients with a central nervous system (CNS) tumor. G enomic DNA was isolated from a small number of cells harvested from the spinal fluid of the patients. The DNA was amplified by DOP-PCR and analyzed by CG H as described in the text. CG H reveals a strong amplification signal on both homologues of chromosome 8q (arrows). The image was photographed directly using a conventional camera on an epifluorescence microscope.
The D O P-PC R and C G H procedures described in this article were used to detect amplified sequences in genomic D N A from spinal fluid of patients who have a tumor of the central nervous system (Figure 4). D O P-PC R and C G H also revealed an amplification of the c-myc oncogene (which maps to chromosomal band 8q24) in cell line H L60 (Figure 5).
Figure 5: Detection of amplified
c-myc oncogene sequences in cell line HL60. G enomic DNA (equivalent to the DNA from only 3 4 HL60 cells) was diluted in TE buffer, amplified by DOP-PCR, and analyzed by CG Has described in the text. After CG H, the amplification signal is clearly visible on both chromosome 8 homologues (arrows). The image was captured as a gray scale image using a CCD camera (Photometrix), pseudocolored, and photographed directly from the computer screen.
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Reagents available from Boehringer Mannheim for these procedures

Product DOP-PCR Master* , ** Description for 25 amplification reactions and 5 control reactions Cat. No. 1 644 963
The D O P-PCR Master contains:

Reagent DOP-PCR mix, 2 x concentrated Description 25 units Taq DNA Polymerase/500 l; 3 mM MgCl2; dATP , dG TP , dCTP , dTTP , 0.4 mM each; 100 mM KCl; 20 mM Tris-HCl; 0.01% (v/v) Brij 35; pH 8.3 (20 C) Available as Three vials No. 1, 3 x 500 l One vial No. 2, 150 l
DOP-PCR primer (Sequence, 40 M in double distilled H2O 5'- CCG ACT CGA GNN NNN NAT GTG G-3' (N = A, G, C, or T, in approximately equal proportions) Double distilled H2O , PCR grade Control DNA Two vials No. 3, Human genomic DNA from the lymphoblastoid cell line BJA, 1 ng/l, in double distilled H2O
2 x 1000 l One vial No. 4, 50 l
O ther reagents:
Reagent DIG-Nick Translation Mix* for in situ probes Description Cat. No. Pack size 160 l
5x conc. stabilized reaction buffer in 50% 1745 816 glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mMdATP , 0.25 mMdCTP , 0.25 mMdG TP , 0.17 mM dTTP and 0.08 mM DIG -11-dUTP . 5x conc. stabilized reaction buffer in 50% 1745 824 glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mM dATP , 0.25 mM dCTP , 0.25 mM dG TP 0.17 mM dTTP and 0.08 mM biotin-16-dUTP . 1 428 578 1 207 750
5
**This product or the use of this product may be covered by one or more patents of Boehringer Mannheim G mbH, including the following: EP patent 0 649 909 (application pending). **This product is sold under licensing arrangements with Roche Molecular Systems and The Perkin-Elmer Corporation. Purchase of this product is accompanied by a license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with an Authorized Thermal Cycler. For complete license disclaimer, see inside back cover page + .
Biotin-Nick Translation Mix* for in situ probes
160 l
Streptavidin-Fluorescein Anti-Digoxigenin-Rhodamine
1 mg 200 g
Product High Pure PCR Product Purification Kit**
Description Kit for 50 purifications Kit for 250 purifications
Cat. No. 1732668 1732676
The H igh Pure PCR Product Purification Kit contains:

Reagent Binding buffer, green cap Wash buffer, blue cap Description Available as Vial 1 Vial 2
nucleic acids binding buffer; 3 M guanidine-thiocyanate, 10 mM Tris-HCl, 5% (v/v) ethanol, pH 6.6 (25 C) wash buffer; add 4 volumes of absolute ethanol before use! Final concentrations; 20 mM NaCl, 2 mM Tris-HCl, pH 7.5 (25 C), 80% ethanol Elution buffer elution buffer; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5 (25 C) High Pure filter tubes Polypropylene tubes, containing two layers of a specially pre-treated glass fibre fleece; maximum sample volume: 700 l Collection tubes 2 ml polypropylene tubes
Vial 3
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T roubleshooting CG H experiments
Control to detect CGH of insufficient quality H ybridization of a D O P-PC R probe to normal human male metaphase chromosomes should show a markedly weaker staining of the X chromosome than of other chromosomes. If there is not a marked difference between the X chromosome and other chromosomes, the control indicates a defective or poor quality C G H experiment. U sually these poor hybridization results are accompanied by other hallmarks of poor quality, like a speckled, non-homogeneous staining pattern or high background fluorescence, both of which may considerably disturb the quantitative image analysis by causing high variability within the ratio profiles. Such poor hybridization results can be due to many causes. Several are listed below. Potential problems with chromosomal preparations In our experience, the most important factor for good hybridization experiments is the careful preparation of metaphase spreads. Therefore, every new batch of chromosomes needs to be tested. If good hybridization results are not obtained, discard the whole batch and start a new slide preparation procedure. N ote: A lthough im paired hybridiz ation is m ainly due to residual cell debris on the slides, proteolytic digestion of the chrom osom es w ill not lead to high quality C G H experim ents. A lthough proteolytic digestion w ill im prov e the hybridiz ation pattern on suboptim al slides, the results are considerably w orse than those obtained on optim um slides w hich hav e not been pretreated w ith protease. Potential problems with probes Two important causes for granular and non-homogeneous staining patterns are: Probe D N A preparations may be contaminated with high amounts of protein. The size of the labeled probe fragments after nick translation may be inappropriate. Fragments which are too large
typically result in a starry background outside of the chromosomes. In contrast, probes which are too small produce a homogeneous background distributed over all chromosomes. Smaller probes may also lead to a homogeneously strong staining pattern in regions which are over- or under-represented in the tumor genome. Problems which may hinder evaluation of CGH In contrast to conventional FISH experiments, strong fluorescence on the chromosomes and little or no fluorescence in the areas where no chromosomes or nuclei are located, are no guarantee that a hybridization experiment will be useful for a C G H analysis. Even in cases where a bright and smooth staining is observed, other factors can severely impair evaluation of the hybridization experiment. Such impairment has two principal causes: Insufficient suppression of repetitive sequences may lead to incorrect measurements of ratios in chromosomal regions which contain many highly variable repetitive sequences, e.g., sequences on chromosome 19. Strong staining of the heterochromatin blocks on chromosomes 1, 9, 16, and 19 can serve as an indicator for poor suppression. N ote: G ood suppression w ill lead to v ery low FI T C and rhodam ine fluorescence intensities in the heterochrom atic chrom osom e regions. C onsequently, ev en v ery sm all v ariations of fluorescence intensities m ay cause gross ratio v ariations w ithin these chrom osom al regions. T hus, they are excluded from ev aluation. Remedy: Either increase the amount of the C o t-1 D N A fraction or use longer preannealing times (the latter being less effective) to achieve adequate suppression of signals in such regions of the genome.
N on-homogeneous illumination of the
optical field leads to considerable regional variations within the ratio image. Such variations make a quantitative evaluation of the C G H experiment completely impossible. Remedy: C enter the light source of the microscope carefully.
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Problems inherent in sample composition Apart from the experimental factors listed above, the validity of a C G H experiment critically depends on the percentage of cells carrying chromosomal imbalances. If no gains or losses of chromosomes are detected, the sample may contain only a low proportion of tumor cells. Therefore, adequate information from the histological laboratory is crucial for any type of C G H analysis.
References
Bohlander, S. K.; Espinosa, R.; Le Beau, M. M.; Rowley, J. D.; Diaz, M. O. (1992) A method for the rapid sequence-independent amplification of enomics microdissected chromosomal material. G 13, 13221324. Du Manoir, S.; Schrck, E.; Bentz, M.; Speicher, M. R.; Joos, S.; Ried, T .; Lichter, P .; Cremer, T . (1995) Quantitative analysis of comparative genomic hybridization. Cytometry 19, 2741. Du Manoir, S.; Speicher, M. R.; Joos, S.; Schrck, E.; Popp, S.; Dhner, H.; Kovacs, G .; Robert-Nicoud, M.; Lichter, P .; Cremer, T . (1993) Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum. G enet. 90, 590610. Joos, S.; Scherthan, H.; Speicher, M. R.; Schlegel, J.; Cremer, T .; Lichter, P . (1993) Detection of amplified genomic sequences by reverse chromosome painting using genomic tumor DNA as probe. Hum. G enet. 90, 584589. Kallioniemi, A.; Kallioniemi, O.-P .; Sudar, D.; Rutovitz, D.; G ray, J. W .; W aldman, F .; Pinkel, D. (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, 818821. Kallioniemi, O-P .; Kallioniemi, A; Piper, J.; Isola, J.; W aldman, F .; G ray, J. W .; Pinkel, D. (1994) Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in enes, Chromosomes and Cancer 10, solid tumors. G 231243. Kallioniemi, O.-P .; Kallioniemi, A.; Sudar, D.; Rutovitz, D.; G ray, J. W .; W aldman, F .; Pinkel, D. (1993) Comparative genomic hybridization: a rapid new method for detecting and mapping DNA amplification in tumors. Seminars in Cancer Biology 4, 4146. Kawasaki, E. S. (1990) Sample preparation from blood, cells, and other fluids. In: Innis, M. A.; G elfand, D. H.; Sninsky, J. J.; White, T . J. (eds) PCR Protocols: AG uide to Methods and Applications. San Diego, CA: Academic Press, 146152. Lichter, P .; Bentz, M.; du Manoir, S.; Joos, S. (1994) Comparative genomic hybridization. In: Verma, R; Babu S. (eds) Human Chromosomes. New Y ork: McG raw Hill, 191210. Lichter, P .; Cremer, T . Chromosome analysis by non-isotopic in situ hybridization. In: Human Cytogenetics: Constitutional Analysis. IRL Press. Lichter, P .; Tang, C. C.; Call, K.; Hermanson, G .; Evans, G . A.; Housman, D.; W ard, D. C. (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 6469. Lundsteen, C.; Maahr, J.; Christensen, B.; Bryndorf, T .; Bentz, M.; Lichter, P .; G erdes, T . (1995) Image analysis in comparative genomic hybridization. Cytometry 19, 4250. Maniatis, T .; Fritsch, E. F .; Sambrook, J. (1986) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New Y ork: Cold Spring Harbor Laboratory Press. Piper, J.; Rutovitz, D.; Sudar, D.; Kallioniemi, A.; Kallioniemi, O.-P .; W aldmann, F . M.; G ray, J. W .; Pinkel, D. (1995) Computer image analysis of comparative genomic hybridization. Cytometry 19, 1026. Speicher, M. R.; du Manoir, S.; Schrck, E.; HoltgreveG rez, H.; Schoell, B.; Lengauer, C.; Cremer, T .; Ried, T . (1993) Molecular cytogenetic analysis of formalin-fixed, paraffin-embedded solid tumors by comparative genomic hybridization after universal enet. 2, 19071914. DNA amplification. Hum. Mol. G Telenius, H.; Pelmear, A. H.; Tunnacliffe, A.; Carter, N. P .; Behmel, A.; Ferguson-Smith, M. A.; Nordenskjld, M.; Pfragner, R. (1992) Ponder BAJP: Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. G enes, Chromosomes and Cancer 4, 257263.
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Identificationof chromosomesinmetaphasespreads and interphase nuclei with PRINS oligonucleotide primersandDIG - or rhodamine-labeled nucleotides
D r. R . Seibl, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any. PRimed I N Situ Labeling (PRIN S) is a fast alternative to traditional in situ hybridization (Koch et al., 1989). PRIN S starts with in situ hybridization (annealing) of an unlabeled synthetic oligonucleotide (e.g., one of the PRIN S oligonucleotide primers in Table 1) to denatured chromosomes in metaphase spreads or interphase nuclei on microscope slides. Then, the hybridized oligonucleotide serves as primer which a thermostable D N A polymerase (G osden et al., 1991) elongates in situ . D uring the primer elongation reaction, the polymerase incorporates nonradioactively labeled nucleotides into the newly synthesized D N A (G osden and H anratty, 1993). D epending on the label, the newly synthesized D N A may be detected by one of two methods: Indirect detection using the D IG PRIN S Reaction Set or direct detection using the Rhodamine PRIN S Reaction Set.
I ndirect detection uses a fluorochrome-
optimal fluorochrome for the PRIN S reaction with the convenience of the PRIN S reaction. This combination produces extremely rapid results as there is no conjugate incubation step required and the best sensitivity in direct detection comparable to indirect detection. In the Rhodamine PRIN S Reaction Set, dTTP is provided at an optimized concentration in the Rhodamine Labeling Mix because dTTP variation has only minor influence on rhodamine labeling intensity.
Table 1: Specificity of PRINS oligonucleotide primers.
PRIN S oligonucleotide primer 1 3 7 8 9 (Sat) 10 11 12 14 + 22 1 + 16
Specific for
Satellite II D N A in the pericentromeric heterochromatin on the long arm of human chromosome 1 C entromere of human chromosome 3, Alpha satellite C entromere of human chromosome 7, Alpha satellite C entromere of human chromosome 8, Alpha satellite Satellite III D N A in the pericentric heterochromatin on the proximal long arm of human chromosome 9 C entromere of human chromosome 10, Alpha satellite C entromere of human chromosome 11, Alpha satellite C entromere of human chromosome 12, Alpha satellite C entromeres of human chromosomes 14 and 22, Alpha satellite Satellite II D N A of human chromosomes 1 and 16 in both cases located to the proximal long arm of that chromosome. C entromere of human chromosome 17, Alpha satellite C entromere of human chromosome 18, Alpha satellite C entromere of human chromosome X, Alpha satellite Long arm of human chromosome Y. Stains most of the long arm of the Y-chromosome. Telomeres of all human chromosomes1 All human chromosomes
conjugated anti-D IG antibody (for example, Anti-D igoxigenin-Fluorescein from the D IG PRIN S Reaction Set) to locate digoxigenin-(D IG -)labeled D N A. For indirect detection, we offer the D IG PRIN S Reaction Set, which combines the high sensitivity of digoxigenin labeling with the power of the PRIN S reaction. In the D igoxigenin PRIN S Reaction Set the sensitivity of the PRIN S reaction can be optimized by adjusting the dTTP-concentration individually according to experimental requirements.
D irect detection
17 18 X Y T Control primer
uses fluorochromelabeled nucleotides (e.g. RhodaminedU TP from the Rhodamine-PRIN S Reaction Set) that are directly visible under a fluorescence microscope. For direct detection, we offer the Rhodamine PRIN S Reaction Set, which combines the labeling intensity of our improved rhodamine which is the
1 Please note the Telomere Primer is only for use with the Digoxigenin PRINS Reaction Set, because the Rhodamine PRINS Reaction Set contains dTTP in the Rhodamine Labeling Mix.
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C ompared to traditional ISH methods (where probe labeling is performed before the probe is added to the slide), PRIN S has two advantages:
C onv enience. PRIN S circumvents the
I. Preparation of slides
1 U sing standard methods (e.g. G osden,
laborious probe isolation and labeling needed in other approaches.

Speed. Since high probe concentrations
are possible, hybridization and chain elongation (in one reaction) usually takes 30 min. For many PRIN S primer the reaction time can even be reduced. The complete procedure, including fluorescent antibody detection, is complete in approximately 2 h. When using the Rhodamine PRIN S Reaction Set for direct detection results are already available after 60 min. This article describes the use of the PRIN S Reaction Set, the Rhodamine-PRIN S Reaction Set, and PRIN S oligonucleotide primers to rapidly identify human chromosomes. It also details how to combine PRIN S and FISH (fluorescence in situ hybridization) for a multiple labeling experiment.
1994; Roonea and C zepulkowski, 1992), prepare fresh microscope slides containing fixed metaphase chromosomes and/ or interphase nuclei. C aution: For best results, prepare the slides containing the fixed m etaphase chrom osom es either im m ediately before use or on the day before the PR I N S reaction. I f slides hav e been prepared a day early, store them at 4 C until use. Such slides m ay be used in the PR I N S reaction w ithout form am ide pretreatm ent (Step 2 below ).
2 (O ptional) If slides have been stored for
3 or more days at room temperature in 70% ethanol at 4C , denature the preparations with formamide directly before the PRIN S reaction. For the formamide treatment, perform the following steps: Prepare a solution containing: 70 ml deionized formamide 10 ml 20 x SSC 10 ml 500 mM sodium phosphate buffer, pH 7.0 10 ml H 2O Adjust the pH of the formamide solution to 7.0 with H C l. Incubate the slides for 45 s at 68 C in the formamide solution. Check the temperature of the formamide solution, as it is important that it be 68C. Wash the slides in a series of ice-cold ethanol solutions (70% , then 90% , then 100% ethanol). C aution: T he ethanol solutions m ust be ice-cold during this step.
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II. Preparation of PRINS reaction mixes

N ote: For a single reaction under a 22 x 22 m m cov erslip, prepare 30 l of D I G PR I N S reaction m ix or R hodam ine-PR I N S reaction m ix. A djust the v olum e of the reaction m ix (but not the concentration of the com ponents) if you are preparing m ore than one reaction, or if you use cov erslips of a different siz e. For 18 x 18 m m cov erslips, prepare 15 m l reaction m ix per reaction; for 24 x 60 m m or 22 x 40 m m cov erslips, prepare 60 m l per reaction.
1 D epending on the type of PRIN S T he dT T P concentration can be
adjusted indiv idually to increase the signal strength by reducing the dT T P concentration or to decrease the signal strength by increasing the dT T P concentration.
2.5 l Taq DN A Polymerase (1 unit/l).
reaction desired, do one of the following: A. I f you want to use D igoxigenin for in situ labeling and detect the PRIN S target by indirect fluorescence (immunological) methods, prepare 30 l of D IG -PRIN S reaction mix (for each reaction) by adding the following components to a sterile microcentrifuge tube (on ice): N ote: M ost of the com ponents (num bered v ials) used in the D I G -PR I N S reaction m ix are from the PR I N S R eaction Set.
13.5 l sterile redistilled H 2O . 3.0 l 10 x concentrated PRIN S 3.0 l 10 x concentrated D IG -PRIN S
B. I f you want to use rhodamine-labeled D N A and detect the PRIN S target by direct fluorescence methods, prepare 30 l of Rhodamine-PRIN S reaction mix (for each reaction) by adding the following components to a sterile microcentrifuge tube (on ice): N ote: M ost of the com ponents (num bered v ials) used in the R hodam inePR I N S reaction m ix are from the R hodam ine-PR I N S R eaction Set.
16.5 l sterile redistilled H 2O . 3.0 l 10 x concentrated PRIN S 3.0 l 10 x concentrated Rhodamine-
reaction buffer (Rh-PRIN S vial 2).
reaction buffer (PRIN S vial 3).
labeling mix (500 M each of dATP, dC TP, and dG TP; 50 M D IG dU TP; 50% glycerol) (PRIN S vial 1). 5.0 l PRIN S oligonucleotide primer [either control primer (PRIN S vial 4) or a human chromosome-specific PRIN S oligonucleotide primer (Table 1). 3.0 l 450 mM dTTP (PRIN S vial 2). C aution: T he concentration of dT T P used in the D I G -PR I N S reaction m ix depends upon the PR I N S oligonucleotide prim er used. U sing the PR I N S O ligonucleotide Prim er T, no -dT T P has to be added to the reaction m ix. For the PRIN S O ligonucleotide Primer 9, the final concentration of dTTP in the DIG-PRIN S reaction mix must be 90 M. For all other PR I N S prim ers listed in Table 1 the final concentration of dT T P in the D I G -PR I N S reaction m ix m ust be 45 M . T he correct dT T P concentration for other prim ers m ust be determ ined em pirically.
PRIN S labeling mix (500 M each of dATP, dC TP, and dG TP; 50 M dTTP; 10 M rhodamine-dU TP; 50% glycerol) (Rh-PRIN S vial 1). Note: The correct dTTP concentration (5 M) for most primers is provided as part of the Rhodamine-PRIN S labeling mix (in the RhodaminePRIN S Reaction Set). Variations in dTTP do not significantly affect the intensity of rhodamine labeling. Since dTTP is included in the Rhodamine-PRIN S labeling mix and because for the PRIN S O ligonucleotide Primer T, no dTTP has to be added to the reaction mix, the T primer is incompatible with the components of the Rhodamine-PRIN S Reaction Set.
5.0 l PRIN S oligonucleotide primer
[either control primer (Rh-PRIN S vial 3) or a human chromosomespecific PRIN S oligonucleotide primer (Table 1).
2.5 l Taq DN A Polymerase (1 unit/l). 2 G ently vortex the reaction solution to 3 C entrifuge the reaction mixture briefly
mix the components.
to collect the liquid on the bottom of the tube.
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III. Primer annealing and elongation

1 Place the dry slides containing fixed I f slides are 3 or more days old and
chromosomes and/ or interphase nuclei on either a heating block or a special thermal cycler for slides. N ote: To guarantee specificity and sensitiv ity of the reaction, the denaturation step and the annealing/ elongation step need precisely controlled elev ated tem peratures. For reproducible results, w e recom m end that, instead of a heating block , you use a tem perature cycler that is specially designed for slides, such as the O m niSlide or O m niG ene Tem perature C ycler (H ybaid L td., Teddington, M iddlesex, England) or an equiv alent instrum ent from another m anufacturer.
2 D epending on the age of the slides, do
have been treated with formamide, skip this step, leave the heating block set at 60C and go to Step 5. 5 Reduce the temperature of the heating block to 60 C and incubate the slides for 30 min at 60C while the primer anneals and the polymerase elongates it. C aution: Be sure the tem perature of the slides is exactly as indicated for the indiv idual prim ers. Ev en a few seconds of exposure to a tem perature below the stringent annealing tem perature can lead to an unw anted back ground signal. T he slides are usually at the correct tem perature w hen the surface of the heating block reaches 60C . Note: The stringent annealing/ elongation temperature for the PRIN S reaction depends on the sequence of the primer used. T he annealing tem perature in the PR I N S reaction using the O ligonucleotide Prim er T is 60C . T he annealing tem perature for all other PR I N S O ligonucleotide Prim er in the PR I N S reaction is 6065C .
6 Remove the slides from the heating
one of the following: I f slides are freshly prepared (one day old or less), incubate them at 94 C for 1 min. I f slides are 3 or more days old and have been treated with formamide (Procedure I, Step 2 above), incubate them at 60C for 1 min. 3 Without removing the slides from the heating block, do the following: O nto each sample, immediately pipette: 25 l of D IG -PRIN S reaction mix or 27 l of Rhodamine-PRIN S reaction mix C over sample with a 22 x 22 mm coverslip. C aution: I f you use cov erslips of a different siz e than 22 x 22 m m , adjust the v olum e of the D I G -PR I N S or R hodam ine-PR I N S reaction m ix in this step so it cov ers the sam ple w ell and com pletely w ets the cov erslip.
4 D epending on the age of the slides, do
block and place them immediately in a C oplin jar containing stop buffer which has been prewarmed to the temperature of the hybridization/ elongation reaction (60C ). 7 Stop the reaction by washing the slides for 35 min at the temperature of the hybridization/ elongation reaction (60 C ) with prewarmed stop buffer. N ote: C ov erslips should drop off the slides during this step. Caution: Be sure the temperature remains the temperature of the hybridization/ elongation reaction (60C) throughout the wash.
one of the following: I f slides are freshly prepared (1 day old or less), incubate them at 9194C for 3 min to denature the chromosomal D N A, then go to Step 5. C aution: T he slides generally reach 9194 C w hen the surface of a heating block reaches 9497 C . H ow ev er, if you are using a tem perature cycler designed for slides, the cycler may autom atically tak e into account this tem perature differential.
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IV . Detection of hybridized sequences

1 In a C oplin jar, wash the slides as follows: 3 x 5 min at 37C with wash buffer
V . Combining PRINS and FISH

1 Label a D N A probe with digoxigenin,
[0.2% Tween 20 in phosphate buffered saline (PBS)]. About 1 min with PBS at room temperature. 2 D epending on the label used in the PRIN Sreaction, do one of the following: I f you produced D IG -labeled D N A in Procedure III, go to Step 3. I f you produced rhodamine-labeled D N A in Procedure III, go to Step 6. 3 Prepare the antibody working solution by diluting the Anti-D igoxigeninFluorescein conjugate (PRIN S vial 5) 1:1000 with PBS containing 1% blocking solution. Note: Anti-DIG-Fluorescein conjugate is a included in the PRIN S Reaction Set. 10 x blocking solution is included in the DIG Wash and Block Set*; it contains 100 mM maleic acid (pH 7.5), 150 mM N aCl, and 10% blocking reagent. PBS containing 1% blocking solution can be prepared by diluting the 10 x blocking solution 1:10 with PBS. 4 In a C oplin jar, incubate the slides with antibody working solution for 30 min in the dark at 37 C . 5 Repeat Step 1 washes. 6 For counterstaining, add the counter stain working solution (e.g., PBS containing either 20 ng/ ml propidium iodide or 20 ng/ ml D API) to the C oplin jar and incubate the slides with the counterstain for 5 min in the dark at room temperature. C aution: I f the target D N A is labeled w ith rhodam ine-dU T P, do not use propidium iodide counterstain, since the em ission of propidium iodide and rhodam ine ov erlap. U se D A PI as a counterstain for rhodam ine-labeled D N A . Wash the slides for 23 min under running water. 7 Air-dry the slides in the dark. 8 Prepare the slides for viewing by doing the following: Apply 20 l antifade solution [e.g., Vectashield (Vector) or Slow FadeLight (Molecular Probes)] to each slide. Add a coverslip. N ote: For long term storage of the slides seal the edges of the cov erslip w ith nail polish and store in the dark at 20 C . 9 Analyze the slides under a fluorescence microscope with the appropriate filters.
biotin, or any fluorochrome (except the one used in the PRIN S procedure), according to procedures described in C hapter 4 of this manual. 2 Perform the PRIN S reaction as described in Procedure IIII of this article. 3 After washing the PRIN S slides in stop buffer (Procedure III, Step 6), dehydrate the slides in an ethanol series (increasing concentrations of ice-cold ethanol). C aution: T he ethanol solutions m ust be ice-cold during this step.
4 H ybridize the labeled D N A probe
(from Step 1) to the PRIN S slides, according to FISH procedures described elsewhere (Procedure IIA, IIB, or IIC from the article In situ hybridization to human metaphase chromosomes using D IG -, biotin-, or fluorochrome-labeled D N A probes and detection with fluorochrome conjugates, in C hapter 5 of this manual, pages 62, 63). Modify the hybridization procedure as follows: D enature the labeled probe D N A before adding it to the slides. D o not heat the slides to 80C after adding the probe to the slides. 5 After the incubation step, wash the slides with the formamide/SSC washes described in the FISH procedure, then perform a final wash in PBS. 6 D epending on the label used in the PRIN S reaction, do one of the following: For PRIN S targets labeled with D IG dU TP, perform the PRIN S antibody detection and wash steps as above (this article, Procedure IV, Steps 35). For PRIN S targets labeled with rhodamine-dU TP, skip this step and go to Step 7. 7 D epending on the label used in the FISH procedure, do one of the following: For biotin- or digoxigenin-labeled FISH probes, perform the immunological detection step according to procedures described elsewhere (Procedure IIIA1/ IIIA2 or IIIB1/ IIIB2 from the article In situ hybridization to human metaphase chromosomes using D IG -, biotin-, or fluorochromelabeled D N A probes and detection with fluorochrome conjugates, in C hapter 5 of this manual, page 64). For fluorochrome-labeled FISH probes, skip this step and go to Step 8.
*Sold under the tradename of G enius in the US.
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8 C ounterstain and prepare the slides as
above (this article, Procedure IV, steps 67). 9 Analyze the slides under a fluorescence microscope with the appropriate filters.
VI. T roubleshooting guide for PRINS

U se the following tips to diagnose and correct commonly occurring problems in the PRIN S procedure described above.
A. If signal is weak or missing, then:
Possible cause
Possible solution
D enaturation temperature does not reach Increase the temperature of the block 9194C at the surface of the slide. slightly during denaturation. Annealing/ elongation temperature is too high. Lower the annealing/ elongation temperature by 3C and repeat experiment. If signal is inadequate, reduce the temperature further (in 3C steps). 1. Reduce the dTTP concentration in the reaction solution to 30 M, 15 M or zero by adding 2 l, 1 l, or no dTTP solution (D IG PRIN S Reaction Set vial 2) to each 30 l reaction 1. Increase the amount of primer in the reaction solution. U se the control primer (D IG PRIN S Reaction Set vial 4) to check the sensitivity of the reaction.
(For D IG -labeling only) N ot enough D IG -dU TP was incorporated into sample.
Primer concentration too low
B. If signal is strong, then:
Possible cause
Possible solution 1. Increase the dTTP concentration in the reaction solution to 60 M, 90 M or 135 M by adding 4 l, 6 l, or 9 l dTTP solution (D IG PRIN S Reaction Set vial 2) to each 30 l reaction 1. D ecrease the amount of primer in the reaction solution. U se the control primer (D IG PRIN S Reaction Set vial 4) to check the sensitivity of the reaction.
(For D IG -labeling only) Too much D IG -dU TP was incorporated into sample.
Primer concentration too high
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C. If chromosomes or nuclei contain unwanted background signals, then:
Possible cause The slide was exposed to temperatures below the stringent annealing/ elongation temperature for a few seconds after the denaturation step. Annealing/ elongation temperature is too low.
Possible solution D o not allow the temperature on the slide to ever fall below the stringent annealing/ elongation temperature. Raise the annealing/ elongation temperature by 3C and repeat experiment. If background is still high, increase the temperature further (in 3C steps) until the reaction is specific. Prewarm the stop buffer to the stringent annealing/ elongation temperature and transfer the slides directly from the heating block to the warm stop solution. Perform a control PRIN S reaction without added primer. If you obtain a signal, either block the 3' ends of the chromosomal breaks by incubating the chromosomal preparations with D N A polymerase and a mixture of ddN TPS or (preferably) discard the chromosomal preparations and prepare fresh chromosomes or fresh chromosomal spreads. Increase the dTTP concentration in the reaction solution to 60 M, 90 M or 135 M by adding 4 l, 6 l, or 9 l dTTP solution (D IG PRIN S Reaction Set vial 2) to each 30 l reaction or D ecrease the amount of primer in the reaction solution.
The slide was exposed to temperatures below the stringent annealing/ elongation temperature for a few seconds between the elongation step and the addition of stop buffer. Primer-independent elongation of nicks and chromosomal breaks occurred. Such nicks and breaks can occur during long term storage of the chromosomal preparation.
(For D IG -labeling only) When the primary signal is strong, minor primerdependent signals may be seen at secondary binding sites for the primer.
D. If the entire slide (not just the chromosomes and nuclei) contain unwanted background signals, then:
Possible cause (For D IG -labeling only) The antibody conjugate (anti-digoxigenin-fluorescein) bound nonspecifically to the slide or to cellular proteins and matrix in the chromosomal preparation.
Possible solution Perform a control detection reaction by incubating an unhybridized sample slide directly with the antibody conjugate, then washing and analyzing the slide as in Procedure IV. I f background is observed, either increase the number and duration of the washing steps or (preferably) repeat the chromosomal preparation to remove excess cellular proteins and matrix responsible for nonspecific binding. Perform a control PRIN S reaction in the absence of Taq polymerase and primer. I f background is observed, either increase the number and duration of the washes or (preferably) repeat the chromosomal preparation to remove excess cellular proteins and matrix responsible for nonspecific binding.
D IG -dU TP or rhodamine-dU TP bound nonspecifically during the PRIN S reaction.
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Results
Figure 1: PRINS labeling and indirect fluorescent detection of human chromosomes. The PRINS primers specific for (Panel a) human chromosome 11 and (Panel b) human chromosome 7 were hybridized to human chromosomal preparations. The hybridized primers were elongated in situ and the newly synthesized DNA labeled with DIG -dUTP according to the procedure described in the text. Labeled DNA was visualized with anti-DIG -fluorescein. Propidium iodide was used as a counterstain.
b1
Figure 2: Rhodamine PRINS labeling and direct
fluorescent detection of human chromosomes. The PRINS primers specific for (Panel a) human chromosome 1 and (Panel b1, b2) human chromosome 9 were hybridized to human chromosomal preparations. The hybridized primers were used to label target DNA in situ with rhodamine-dUTP according to the procedure described in the text. Labeled DNA was viewed directly under a fluorescence microscope. DAPI was used as a counterstain.
b2 G osden, J.; Hanratty, D. (1993) PCR in situ: A rapid alternative to in situ hybridization for mapping short, low-copy number sequences without isotopes. BioTechniques 15, 7880. Koch, J. E.; Klvraa, S.; Petersen, K. B.; G regersen, N.; Bolund, L. (1989) Oligonucleotide priming methods for the chromosome-specific labeling of alpha satellite DNA in situ. Chromosoma (Berl.) 98, 259265. Roonea, D. E.; Czepulkowski, B. H., eds. (1992) Human Cytogenetics, A Practical Approach, Vol. 1. Oxford, England: IRL Press.
References
G osden, J. R., ed. (1994) Chromosome Analysis Protocols (Methods Mol. Biol., Vol. 29). Totowa, NJ: Humana Press. G osden, J.; Hanratty, D.; Starling, J.; Fantes, J.; Mitchell, A.; Porteus, D. (1991) Oligonucleotide primed in situ DNA synthesis (PRINS): A method for chromosome mapping, banding and investigation of enet. 57, sequence organization. Cytogenet. Cell G 100104.
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Reagents available from Boehringer Mannheim for PRINS

Product DIGPRINS Reaction Set Description For 40 PRINS labeling reactions and 5 control reactions Cat. No. 1 695 932
The D IG PRIN S Reaction Set includes a detailed working procedure and contains:
Reagent 10 x Concentrated PRINS labeling mix dTTP solution 10 x Concentrated PRINS reaction buffer PRINS Control Oligonucleotide Primer Anti-Digoxigenin-Fluorescein Conjugate, Fab fragments Product Rhodamine PRINS Reaction Set Description dNTPs including digoxigenin-11-dUTP 450 M dTTP Buffer for 45 reactions Primer for 5 control reactions Antibody (200 g/ml) for 22 detection reactions in a Coplin jar Description For 40 PRINS labeling reactions and 5 control reactions Kit component One vial No. 1, 135 l One vial No. 2, 200 l One vial No. 3, 150 l One vial No. 4 Two vials No. 5, 2 x 550 l Cat. No. 1 768 514
The Rhodamine PRIN S Reaction Set includes a detailed working procedure and contains:
Reagent 10 x Concentrated Rhodamine PRINS labeling mix 10 x Concentrated PRINS reaction buffer PRINS Control Oligonucleotide Primer Description dNTPs including Rhodamine-dUTP Buffer for 50 reactions Primer for 5 control reactions Kit component One vial No. 1, 135 l One vial No. 2, 150 l One vial No. 3, 25 l
PRIN S oligonucleotide primers The following PRIN S oligonucleotide primers are available* (Pack size 100 l each):
Primer PRINS oligonucleotide primer 1, specific for human chromosome 1 PRINS oligonucleotide primer 1+16, specific for human chromosome 1+16 PRINS oligonucleotide primer 3, specific for chromosome 3 PRINS oligonucleotide primer 7, specific for human chromosome 7 PRINS oligonucleotide primer 8, specific for human chromosome 8 PRINS oligonucleotide primer 9 (Sat), specific for human chromosome 9 PRINS oligonucleotide primer 10, specific for human chromosome 10 PRINS oligonucleotide primer 11, specific for human chromosome 11 PRINS oligonucleotide primer 12, specific for human chromosome 12 PRINS oligonucleotide primer 14+22, specific for human chromosome 14+22 PRINS oligonucleotide primer 17, specific for human chromosome 17 PRINS oligonucleotide primer 18, specific for human chromosome 18 PRINS oligonucleotide primer X, specific for the human X chromosome PRINS oligonucleotide primer Y , specific for the human Y chromosome PRINS oligonucleotide primer T, specific for human telomeres PRINS control oligonucleotide primer, specific for all human chromosomes Cat. No. 1 695 959 1768 549 1 695 967 1 695 975 1 695 983 1768 565 1768 522 1 695 991 1 696 009 1768 557 1 696 017 1 696 025 1 696 033 1 696 041 1 699 199 1 699 172 * Please note the Telomer Primer is only for use with the Digoxigenin PRINS Reaction Set because the Rhodamine PRINS Reaction Set contains dTTP in the Labeling Mix.
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Identification of chromosomes in metaphase spreads with DIG - or fluorescein-labeled human chromosome-specific satellite DNA probes
D r. G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any.
Fluorescence in situ hybridization (FISH ) with human chromosome-specific satellite D N A probes is a sensitive technique for identifying individual human chromosomes. Applications include: Analysis of aneuploidies in normal or tumor cells Identification of chromosomes in hybrid cells Production of genetic linkage maps Especially useful for such applications are fluorescein- or digoxigenin-labeled probes which recognize alphoid sequence variants that occur at the centromere of specific chromosomes (Willard and Waye, 1987; C hoo, K. H . et al., 1991). Fluoresceinlabeled probes allow rapid, direct detection of a single chromosome. D IG -labeled probes allow sensitive, indirect detection of a chromosome with a variety of fluorochrome-labeled antibodies. Both fluorescein- and D IG -labeled probes can be combined in a multicolor labeling of different chromosomes on a single preparation. This article describes the use of such prelabeled probes to identify human chromosomes in chromosomal spreads from whole blood or from cell culture.
I. Preparation of metaphase chromosomes

1 Prepare chromosomes from either whole For w hole blood, do these steps: Mix 5 ml whole blood with 5 ml PBS. Pipette 7.5 ml of lymphocyte separa-
blood or cell culture as follows:
tion medium into a 50 ml centrifuge tube. C arefully layer blood-PBS mixture atop separation medium. C entrifuge for 30 min at 800 x g. Remove the interphase (middle part of the centrifuged mixture) that contains the lymphocytes. Place the lymphocytes in a fresh centrifuge tube. Mix lymphocytes with 50 ml PBS. C entrifuge for 10 min at 420 x g. D iscard the supernatant and resuspend cells at approximately 106 cells per ml in chromosome medium (G ibco). N ote: T his step should require about 5 m l chrom osom e m edium . G o to Step 2. For cells in culture, do these steps: H arvest cells by centrifugation. Resuspend cells at approximately 106 cells per ml in chromosome medium. G o to Step 2. 2 Incubate cells in chromosome medium for 71 h in a C O 2 incubator at 37C and 7% C O 2. 3 Add 12 l C olcemid per ml medium. 4 Incubate cells for 1 h at 37C and 7% C O 2. 5 C entrifuge cells (210 x g) for 10 min at 4 C . 6 With a pipette, remove supernatant until only 1 ml remains atop the cells.
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7 Resuspend the pellet in the remaining

8 Slowly add to the resuspended cells 10 ml
supernatant.
III. In situ hybridization with chromosome-specific DNA probes

N ote: Perform the follow ing procedure in a 50 m l C oplin jar w hich can hold a m axim um of 8 slides. C aution: Prew arm all solutions in the follow ing procedure to the appropriate tem perature before using them .
1 Remove slides from the 70% ethanol
9 G ently mix and incubate for 1220 min

10 To the mixture, add 7 drops of chilled
of prewarmed (37 C ) 75 mM KC l. at 37 C .
methanol-acetic acid fixative (3 parts methanol to 1 part acetic acid, precooled to 20 C ). G ently mix. 11 C entrifuge mixture (210 x g) for 10 min at 4 C . 12 With a pipette, remove supernatant until only 1 ml remains atop the pellet. 13 C arefully resuspend the pellet in the remaining supernatant and add 1 ml precooled (20C ) methanol-acetic acid fixative. G ently mix. 14 Add an additional 510 ml precooled (20 C ) methanol-acetic acid fixative. Mix thoroughly. 15 Fix chromosomes for at least 30 min at 20 C . 16 Repeat Steps 1115 three to five times. 17 Store the metaphase chromosomes at least 24 h in methanol-acetic acid fixative at 20C . 18 D o either of the following: G o to Procedure II. Store metaphase chromosomes in methanol-acetic acid fixative at 20C up to several months.
II. Preparation of chromosomal spreads

1 Pretreat slides as follows: Wash slides briefly in a 1:1 mixture of Air dry slides. Wash briefly in redistilled H 2O . (D o
2 Centrifuge metaphase chromosomes from
100% ethanol and ether.
not dry.)
3 Remove and discard all supernatant. 4 Resuspend chromosomal pellet in 12 ml
Procedure I (210 x g) for 10 min at 4C.
of precooled (20C ) methanol-acetic acid fixative. 5 D rop the metaphase chromosomes onto a moist, pretreated slide from a height of approximately 50 cm. 6 Allow slides to air dry. 7 For optimal metaphase spreads, store slides at least 5 days in 70% ethanol at 4 C . N ote: You m ay store slides up to sev eral w eek s in 70% ethanol at 4 C .
storage solution (from Procedure II, Step 7). 2 Air dry slides completely (approxi mately 30 min). 3 For each slide, prepare 20 ml hybridization solution containing: 1013 l deionized formamide (final concentration: 5065% formamide) Note: Different probes require different concentrations of deionized formamide. See Table 1 for the correct formamide concentration to use with each probe. 5 l 4 x concentrated hybridization buffer [8 x SSC ; 40% (v/ v) dextran sulfate; 4 mg/ ml MB-grade D N A (from fish sperm); pH 7.0]. 12 l (2040 ng) human chromosome-specific satellite D N A probe, either fluorescein- or D IG -labeled. Enough redistilled H 2O to make a total volume of 20 l. C aution: Tw enty l is enough hybridiz ation solution if you are using a 24 x 24 m m cov erslip in Step 7 below . I f you use a different siz e cov erslip, adjust the v olum e of the hybridiz ation solution (but not the concentration of the com ponents) accordingly. 4 Preheat a glass tray big enough to hold all slides in a 72C oven. 5 Prewarm a moist chamber to 37C . 6 Pipette 20 l hybridization solution onto each slide, add a 24 x 24 mm coverslip, and seal the coverslip to the slide with rubber cement. 7 Place the prepared slides onto the prewarmed glass tray in the oven and incubate 510 min at 72C to denature D N A. 8 Transfer slides to the prewarmed moist chamber and incubate overnight at 37C .
CONTENTS
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89
IV . Detection of hybridized sequences

IVA. For detection of fluorescein-labeled chromosome-specific probes IVB. For detection of DIG-labeled chromosome-specific probes
1 After hybridization, wash slides in a
N ote: Perform Steps 1 and 2 in a C oplin jar.

1 After hybridization, wash slides ac-
cording to one of the following wash schemes: 15 min at 3048C with 1 x SSC containing 50% formamide, pH 7.0; then : 2 x 5 min at room temperature with 2 x SSC , pH 7.0 or 15 min at 37C with 2 x SSC , pH 7.0. N ote: D ifferent probes require different w ashes. See Table 1 for the correct w ashes to use w ith each probe. 2 For counterstaining, add 50 ml of either propidium iodide solution (100 ng/ ml propidium iodide in PBS) or D API solution (100 ng/ ml D API in PBS) to the slides, then incubate the slides for 25 min in the dark at room temperature. 3 Wash slides 23 min under running water. 4 Air dry the slides in the dark. 5 Apply 20 l antifade solution [e.g., 2% D ABC O (w/ v) in PBS containing 50% glycerol] to each slide. 6 Add a 24 x 24 mm coverslip. N ote: For long term storage of the slides, seal the edges of the cov erslip w ith nail polish and store in the dark at 20 C . 7 Analyze the slides under a fluorescence microscope with the appropriate filters. N ote: M axim um em ission w av elength for fluorescein (FL U O S) is 523 nm ; for propidium iodide, 610 nm ; and for D A PI , 470 nm .
C oplin jar according to one of the following wash schemes: 15 min at 4048C with 1 x SSC containing 50% formamide, pH 7.0; then : 2 x 5 min at room temperature with 2 x SSC , pH 7.0 or 15 min at 37C with 2 x SSC , pH 7.0. N ote: D ifferent probes require different w ashes. See Table 1 for the correct w ashes to use w ith each probe. 2 (O ptional) Incubate slides for 30 min at 37 C with 50 ml PBS containing 1% blocking reagent. 3 Just before use, prepare 100 l of a 1 g/ ml working solution of anti-D IG -fluorophore conjugate in PBS containing 1% blocking reagent. N ote: Each slide to be analyz ed requires 100 l of antibody w ork ing solution. N ote: A nti-D I G -fluorophore conjugate can be Anti-DIG-Fluorescein, Anti-DIGA M C A , or A nti-D I G -R hodam ine. 4 Add 100 l freshly prepared antibody working solution to each slide and cover the complete slide with a coverslip. 5 Incubate slides for 30 min at 37C in a moist chamber. 6 In a C oplin jar, wash slides for 3 x 5 min at room temperature with 2 x SSC containing 0.1% Tween 20. 7 C ounterstain, mount and view slides as for fluorescein-labeled probes (Steps 27, Procedure IVA). Exception: For experim ents using antiD I G -rhodam ine conjugates, use D A PI rather than propidium iodide as a counterstain. T he em ission w av elength of propidium iodide is too near that of rhodam ine. N ote: M axim um em ission w av elength for A M C A is 474 nm ; for rhodam ine, 570 nm .
90
CONTENTS
INDEX
Probe1 for chromosomes 1 2 3 4 5 + 1 + 19 6 7 8 9 10 11 12 13 + 21 14 + 22 15 16 17 18 20 22 X Y All human
Labeled with D IG 2 FLU O S D IG FLU O S D IG D IG D IG FLU O S D IG D IG FLU O S D IG FLU O S D IG D IG FLU O S D IG FLU O S D IG FLU O S D IG D IG FLU O S D IG D IG D IG FLU O S D IG FLU O S D IG FLU O S D IG D IG FLU O S D IG FLU O S D IG FLU O S
Formamide (%) in hybridization solution 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 65% 50% 50% 50% 50%
Posthybridization wash conditions Wash 1 (15 min) Wash 2 (2 x 5 min) 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 48C ; 1x SSC + 50% formamide 48C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 48C ; 1x SSC + 50% formamide 48C ; 1x SSC + 50% formamide 30C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 48C ; 1x SSC + 50% formamide 43C ; 1x SSC + 50% formamide 40C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 46C ; 1x SSC + 50% formamide 37C ; 2 x SSC 37 C ; 2 x SSC 37C ; 2 x SSC 37 C ; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC RT; 2 x SSC O mit O mit O mit O mit
1 The probes all recognize a pattern of alphoid DNA on specific chromosomes, except for the chromosome 1 probe (which recognizes satellite III DNA on chromosome 1) and the chromosome Y probe (which recognizes a repeat on the long arm of chromosome Y). 2 Abbreviations used: DIG , digoxigenin; FLUOS, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; 1xSSC, buffer (pH 7.0) containing 150 mM NaCl and 15 mM sodium citrate; RT , room temperature.
Table 1: Hybridization parameters for human chromosome-specific satellite DNA probes. Use the data given in this table in Procedures III and IV of this article. All probes are available from Boehringer Mannheim.
CONTENTS
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91
Results
Figure 1: FISH of human chromosome-specific
satellite DNA probes to metaphase spreads. Fluorescence signals have been pseudocolored with a CCD camera and a digital imaging system. In Panel a, a DIG -labeled probe specific for chromosomes 5 + 1 + 19 was detected with antiDIG -rhodamine. Chromosomes were counterstained with DAPI. In Panel b, a DIG -labeled probe specific for chromosome 12 was detected with anti-DIG fluorescein. Chromosomes were counterstained with propidium iodide. Panel c shows the direct detection of chromosome 12 sequences with a fluorescein-labeled probe. Chromosomes were counterstained with propidium iodide.
References
W aye, J. S.; Willard, H. F . (1987) Nucleotide sequence heterogeneity of alpha satellite repetitive DNA: a survey of alphoid sequences from different human chromosomes. Nucl. Acids Res. 15, 75497569. Choo, K. H.; Vissel, B.; Nagy, A.; Earle, E.; Kalitsis, P . (1991) A survey of the genomic distribution of alpha satellite DNA on all the human chromosomes, and derivation of a new consensus sequence. Nucl. Acids Res. 19, 11791182.
92
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Reagents available from Boehringer Mannheim for this procedure

Reagent DNA, MB-grade from fish sperm DAPI Anti-DigoxigeninFluorescein Anti-DigoxigeninRhodamine Anti-DigoxigeninAMCA Description It prevents unspecific hybridization in all types of membrane or in situ hybridization Fluorescence dye for staining of chromosomes Fab Fragments from sheep Fab Fragments from sheep Fab Fragments from sheep Cat. No. 1 467 140 236 276 1 207 741 1 207 750 1 533 878 Pack size 500 mg (50 ml) 10 mg 200 g 200 g 200 g
These D N A probes are plasmids containing human satellite D N As. C lones were isolated from human-hamster hybrid cell lines containing the respective human chromosomes as the only human compoReagent
nent. The fragment length distribution of the labeled probes shows a maximum at 200500 bases. 1 g probe is sufficient for 2050 hybridizations on 24 x 24 mm coverslips.
Cat. No. 1558 765 1558 684 1690 507 1690 647 1666 479 1666 380 1690 515 1690 523 1690 531 1690 639 1694 375 1666 487 1666 398 1690 540 1690 558 1690 655 1690 566 1690 663 1690 574 1690 671 1690 582 1666 495 1666 401 1690 604 1699 091 1666 452 1666 371 1666 509 1666 428 1666 517 1666 436 1690 612 1666 525 1666 444 1558 196 1558 692 1558 757 1558 676 Pack size 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l) 1 g (50 l)
DNA probes, human chromosome 1 specific, digoxigenin-labeled 1 specific, fluorescein-labeled 1 + 5 + 19 specific, digoxigenin-labeled 1 + 5 + 19 specific, fluorescein-labeled 2 specific, digoxigenin-labeled 2 specific, fluorescein-labeled 3 specific, digoxigenin-labeled 4 specific, digoxigenin-labeled 6 specific, digoxigenin-labeled 7 specific, digoxigenin-labeled 7 specific, fluorescein-labeled 8 specific, digoxigenin-labeled 8 specific, fluorescein-labeled 9 specific, digoxigenin-labeled 10 specific, digoxigenin-labeled 10 specific, fluorescein-labeled 11 specific, digoxigenin-labeled 11 specific, fluorescein-labeled 12 specific, digoxigenin-labeled 12 specific, fluorescein-labeled 13 + 21 specific, digoxigenin-labeled 14 + 22 specific, digoxigenin-labeled 14 + 22 specific, fluorescein-labeled 15 specific, digoxigenin-labeled 16 specific, digoxigenin-labeled 17 specific, digoxigenin-labeled 17 specific, fluorescein-labeled 18 specific, digoxigenin-labeled 18 specific, fluorescein-labeled 20 specific, digoxigenin-labeled 20 specific, fluorescein-labeled 22 specific, digoxigenin-labeled X specific, digoxigenin-labeled X specific, fluorescein-labeled Y specific, digoxigenin-labeled Y specific, fluorescein-labeled Specific for all human chromosomes, digoxigenin-labeled Specific for all human chromosomes, fluorescein-labeled
CONTENTS
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93
Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension

D . C eleda1, 2, U . Bettag1, and C . C rem er1
1 2
I nstitute for A pplied Physics, U niv ersity of H eidelberg. I nstitute for H um an G enetics and A nthropology, U niv ersity of H eidelberg, G erm any.
Fluorescence in situ hybridization (FISH ) has found widespread application in the analysis of chromosomes and interphase nuclei fixed on slides (Anastasi et al., 1990; C remer et al., 1988, 1990; D ekken et al., 1990; D evilee et al., 1988; Kolluri et al., 1990; Lichter et al., 1991; Pinkel et al., 1988; Schardin et al., 1985; Wienberg et al., 1990). H owever, hybridization of specific D N A probes to isolated metaphase chromosomes in suspension offers a new approach to chromosome analysis and chromosome separation. Initial investigations were done on chromosomes obtained from a (C hinese hamster X human) hybrid cell line with biotinylated human genomic D N A as the probe (D udin et al., 1987, 1988; H ausmann et al., 1991). So far the technique for FISH in suspension has been a modification of FISH techniques used for metaphase chromosomes and interphase nuclei fixed on slides. Formamide (and to some extent dextran sulfate) are obligatory components of this method. Thus, the technique requires a certain number of washing steps after hybridization. The washing steps for FISH in suspension, however, are based on centrifugal steps. These steps are responsible for a considerable reduction in the final amount of chromosomal material. Additionally, the existing method for FISH in suspension appears to favor an aggregation of the chromosomal material in suspension. We report here a hybridization technique which does not need formamide and dextran sulfate. As a model system, we used the repetitive specific human D N A probe pU C 1.77 (C ooke and H indley, 1979; Emmerich et al., 1989), labeled it with digoxigenin-11-dU TP by nick-translation, and hybridized it to metaphase chromosomes in suspension. These chromosomes were isolated by standard techniques from human lymphocytes. In preliminary experiments, this technique produced a large number of isolated 94
metaphase chromosomes with satisfactory morphology and clear hybridization signals (Figure 1). Adapting the same method and probe to metaphase spreads (Figure 2) allowed us to determine hybridization efficiency.
Procedure
N ote: T he D N A probe pU C 1.77 is a clone of the plasm id v ector pU C 9 that contains a 1.77 k b hum an Eco R I fragm ent. T he inserted D N A w as isolated from hum an satellite D N A fraction I I / I I I . T his insert contains m ainly a tandem ly organiz ed repetitiv e sequence in the region q12 of chrom osom e 1 (C ook e and H indley, 1979; G osden et al., 1981).
1 Label the D N A probe pU C 1.77 with
D igoxigenin-11-dU TP according to standard nick translation procedures (as described in C hapter 4 of this manual). 2 C ultivate human lymphocytes from peripheral blood. 3 Prepare chromosomes from the lymphocytes by standard techniques. 4 Resuspend the chromosomes and inter phase nuclei in methanol/ acetic acid (3 :1) and store at 20C . 5 Perform fluorescence detection with Anti-D IG -Fluorescein, Fab fragments as described (Lichter et al., 1990), with the following modifications: Incubate for approx. 1 h at 37C . After the blocking step, do not use bovine serum albumin in the rest of the procedure. Modify as necessary to allow FISH in suspension. 6 After labeling and FISH procedures, pipette 10 l of the suspension on a slide for microscopic analysis. C ounterstain with 15 M propidium iodide (PI) and 5 M D API. 7 Analyze with an epifluorescence microscope (O rthoplan equipped with a Planapo oil 63 x objective and a 1.40 aperture, Leitz, Wetzlar, G ermany).
CONTENTS
INDEX
8 Photograph with, for instance, Fuji
chrome P1600 D film at a final image magnification of about 630 x, using Leitz filter system I 2/ 3 for detection of FITC fluorescence.
Results
Figure 1: FISH of digoxigenin-labeled DNA probe pUC 1.77 to isolated human chromosomes in suspension. For details of the hybridization procedure, see the text. The sites of hybridization on the chromosomes appear as yellowish-green spots in the centromeric regions. Counterstaining was with propidium iodide (PI) and DAPI.
5
Figure 2: FISH of a human metaphase spread according to the same hybridization technique used in Figure 1. In this case the pUC 1.77 DNA probe binds to major hybridization sites (q12 region of human chromosome 1) that appear as two large yellowish-green spots on two of the largest chromosomes (large arrowheads). Additionally, a minor binding site of the DNA probe (chromosome 16; G osden et al., 1981) is indicated by two minor yellowish-green spots on two of the smaller chromosomes (small arrowheads).
CONTENTS
INDEX
95

Reagent DIG-Nick Translation Mix* for in situ probes *This product or the use of this product may be covered by one or more patents of Boehringer Mannheim G mbH, including the following: EP patent 0 649 909 (application pending). Description 5 x conc. stabilized reaction buffer in 50% glycerol (v/v) and DNAPolymerase I, DNase I, 0.25 mM dATP , 0.25 mM dCTP , 0.25 dG TP , 0.17 mM dTTP and 0.08 mM DIG -11-dUTP Fab Fragments from sheep Fluorescence dye for staining of chromosomes Cat. No. 1745 816 Pack size 160 l
Anti-DigoxigeninFluorescein DAPI
1 207 741 236 276
200 g 10 mg
Acknowledgments
We thank Prof. D r. T. C remer, Intitute of H uman G enetics and Anthropology, U niversity of H eidelberg, for providing the plasmid pU C 9 and the human lymphocyte preparations. The work was supported by the D eutsche Forschungsgemeinschaft.
Dudin, G .; Steegmayer, E. W .; Vogt, P .; Schnitzer, H.; Diaz, E.; Howell, K. E.; Cremer, T .; Cremer, C. (1988) Sorting of chromosomes by magnetic separation. Human. G enet. 80, 111116. Emmerich, P .; Loos, P .; Jauch, A.; Hopman, A. H. N.; Wiegant, J.; Higgins, M. J.; White, B. N.; van der Ploeg, M.; Cremer, C.; Cremer, T . (1989) Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography. Exp. Cell Res. 181, 126149. G osden, J. R.; Lawrie, S. S.; Cooke, H.J. (1981) A cloned repeated DNA sequence in human chromosome heteromorphisms. Cytogenet. Cell G enet. 29, 3239. Hausmann, M.; Dudin, G .; Aten, J. A.; Heilig, R.; Diaz, E.; Cremer, C. (1991) Slit scan flow cytometry of isolated chromosomes following fluorescence hybridization: an approach of online screening for specific chromosomes and chromosome translocations. Z. Naturforsch. 46c, 433441. Kolluri, R. V .; Manuelidis, L.; Cremer, T .; Sait, S.; G ezer, S.; Raza, A. (1990) Detection of monosomy 7 in interphase cells for patients with myeloid disorders. Am. J. Hermatol. 33 (2), 117122. Lichter, P .; Boyle, A. L.; Cremer, T .: W ard, D. C. (1991) Analysis of genes and chromosomes by enet. Anal. non-isotopic in situ hybridization. G Techn. Appl. 8, 2435. Lichter, P .; Tang, C. C.; Call, K.; Hermanson, G .; Evans, H. J.; Housman, D.; W ard, D. C. (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 6469. Pinkel, D.; Landegent, J.; Collins, C.; Fuscoe, J.; Segraves, R.; Lucas, J.; G ray, J. W . (1988) Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA 85, 91389142. Schardin, M.; Cremer, T .; Hager, H. D.; Lang, M. (1985) Specific staining of human chromosomes in Chinese hamster X human cell lines demonstrates enet. 71, 281287. interphase territories. Hum. G Wienberg, J.; Jauch, A.; Stanyon, R.; Cremer, T . (1990) Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. G enomics 8, 347350.
References
Anastasi, J.; Le Beau M. M.; Vardiman, J. W .; W estbrook, C. A. (1990) Detection of numerical chromosomal abnormalities in neoplastic hematopoietic cells by in situ hybridization with a chromosome-specific probe. Am. J. Pathol. 136, 131139.
Cooke, H. J.; Hindley, J. (1979) Cloning of human satellite III DNA: different components are in different chromosomes. Nucleic Acids Res. 10, 31773197. Cremer, T .; Lichter, P .; Borden, J.; W ard, D. C.; Manuelidis, L. (1988) Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosomeenet. 80, 235246. specific library probes. Hum. G Cremer, T .; Popp, S.; Emmerich, P .; Lichter, P .; Cremer, C. (1990) Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization. Cytometry 11, 110118. Dekken van, H.; Pizzolo, J. G .; Reuter, V . E.; Melamed, M. R. (1990) Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome specific DNA probes. Cytogenet. Cell G enet. 54, 103107. Devilee, P .; Thierry, R. F .; Kievits, T .; Kolluri, R.; Hopman, A. H. N.; Willard, H. F .; Pearson, P . L.; Cornellisse, C. J. (1988) Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes. Cancer Res. 48, 58255830. Dudin, G .; Cremer, T .; Schardin, M.; Hausmann, M.; Bier, F .; Cremer, C. (1987) A method for nucleic acid hybridization to isolated chromosomes in enet. 76, 290292. suspension. Hum. G
96
CONTENTS
INDEX
III. Hybridization and detection

1 Add 5 l of the hybridization mixture
7 Incubate 30 min with anti-D IG anti8 Wash 5 min in PBS. 9 Blot off excess buffer with paper. 10 Finally, mount the chromosomal prepa
body at 37C under a coverslip.
containing the digoxigenin-labeled probe D N A (from Procedure I) to the chromosome preparation and cover with a coverslip (18 x 18 mm). 2 Seal coverslip with rubber cement. 3 Incubate slides at the appropriate hybridization temperature between 50C and 65C (depending on the probe, AT-content, sequence homology, etc.) for 46 h (for single copy sequences) or for 1 h (for repetitive sequences). N ote: L onger incubations do not m ark edly increase the hybridiz ation signal!
4 Remove rubber cement, then wash off
ration in glycerol-para-phenylenediamine-mixture [1 mg p-phenylenediamine dissolved in 1 ml phosphate buffered saline (1 mM sodium phosphate, pH 8.0; 15 mM N aC l) containing 50% glycerol]. 11 Inspect with a fluorescence microscope with the appropriate set of filters. C omments: I nstead of antibodies labeled w ith fluorescent dyes, any other antibody conjugates (enz ym e conjugated, goldlabeled) can be used. I n the case of biotin-labeled D N A , streptav idin can be used instead of antibodies to detect the hybridiz ed D N A . I hav e tested a num ber of these possibilities, and all w ork v ery w ell (including gold-labeled anti-digoxigenin antibody follow ed by silv er-enhancem ent). H ow ev er, in m y experience, the m ost precise localiz ation is obtained w ith im m unofluorescence. I t seem s to m e that the sensitiv ity is also higher (or at least the signal/ back ground ratio is im prov ed) w ith fluorescently labeled antibodies. A dm ittedly, this m ight be a m atter of personal preference.
the coverslip in 2 x SSC for 25 min at room temperature . 5 Wash the slide in PBS for 2 min. Remove excess buffer from the slide by wiping with soft paper around the chromosome preparation. N ote: D o not let the preparation dry com pletely; this produces back ground.
6 Apply 5 l of a 1:10 diluted solution of
fluorescein- or rhodamine-labeled antiD IG antibody. The antibodies should be diluted in PBS containing 1 mg/ ml bovine serum albumin.
Results
Figure 1: Double in situ hybridization of a polytene chromosome of Chironomus with twodifferent clones from a chromosomal walk within the sex-determining region of Chironomus piger. The two -clones contain genomic DNA fragments which are 60 kbp apart. One clone was labeled with digoxigenin and detected with rhodamine-labeled anti-DIG antibody (red); the second clone was labeled with biotin and detected with anti-biotin antibody and fluorescein-labeled secondary antibody (green). The red and green signals can be seen simultaneously if the fluorescence microscope is equipped with a filter system for simultaneous blue and green excitation (filter no. 513803, Leica, W etzlar). For the hybridization, the two probes are mixed 1:1, and the detection is performed with mixed antibody solutions. The method allows a very clear orientation for a chromosomal walk. W e were able to discriminate the location of two clones which were only approximately 30 kb apart.
98
CONTENTS
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Figure 2: Double hybridization of a single copy clone from the sex-determining region together with a highly repetitive DNA element. The single copy DNA fragment was labeled with digoxigenin and the repetitive element with biotin. The single copy DNA(red) hybridizes to a single band, while the repetitive element (green) produces signals over numerous chromosomal sites throughout the entire chromosome. Hybridization and detection of the hybridized DNA was essentially the same as in Figure 1.
References
Langer-Safer, P . R.; Levine, M.; W ard, D. C. (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. USA 79, 43814385. Schmidt, E. R.; Keyl, H.-G .; Hankeln, T . (1988) In situ localization of two haemoglobin gene clusters in the chromosomes of 13 species of Chironomus. Chromosoma (Berl.) 96, 353359.
Figure 3: Double in situ hybridization of biotinand digoxigenin-labeled DNA probes from two different DNA puffs in the salivary gland polytene chromosome C of Trichosia pubescens (Sciaridae, Diptera). The probes were hybridized simultaneously as described in Figure 1. The two sites of hybridization were detected with rhodamine-labeled anti-DIG and anti-biotin antibodies plus fluorescein-labeled secondary antibody. The two differentially labeled sites are so-called DNA puffs , which are chromosomal regions with a developmentally regulated DNA amplification. The double in situ hybridization method allows very rapid mapping of the chromosomes, even when the banding structure of the chromosome is not analyzed in detail.
5
Cat. No. 1 585 606 Pack size 160 l (40 reactions)

Reagent Description DIG-High Prime* , **, *** Complete reaction mixture for random primed labeling of DNA with DIG -dUTP 5x solution with: 1 mM dATP , dCTP , dG TP (each), 0.65 mM dTTP , 0.35 mM DIG -11-dUTP , alkali-labile; random primer mixture; 1 unit/l Klenow enzyme labeling grade, in reaction buffer, 50% glycerol (v/v) Biotin-High Prime** Complete reaction mixture for random primed labeling of DNA with Biotin-dUTP 5x solution with: 1 mM dATP , dCTP , dG TP (each), 0.65 mM dTTP , 0.35 mM biotin-16-dUTP , random primer mixture, 1 unit/l Klenow enzyme labeling grade, in reaction buffer, 50% glycerol (v/v) RNase, DNase-free, can be used directly without prior heat treatment in any DNA isolation technique Fab Fragments from sheep
1 585 649
100 l (25 reactions)
***EP Patent 0 324 474 granted to and EP Patent application 0 371 262 pending for Boehringer Mannheim G mbH. ***This product or the use of this product may be covered by one or more patents of Boehringer Mannheim G mbH, including the following: EP patent 0 649 909 (application pending). ***Licensed by Institute Pasteur.
RNase, DNase-free
1119 915
500 g (1 ml)
Anti-DigoxigeninRhodamine
1 207 750
200 g
CONTENTS
INDEX
99
A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG -labeled DNA probe
Prof. D r. E. R . Schm idt, I nstitute for G enetics, Johannes G utenberg-U niv ersity of M ainz , G erm any.
The protocol given here is a derivative of several published methods (Langer-Safer et al., 1982; Schmidt et al., 1988) with some minor modifications that make the method of in situ hybridization easier, faster, more reliable, and available to anyone who can operate a microscope.
II. Preparation and denaturation of polytene chromosomes from Drosophila, Chironomus, or other species
Squash larval salivary glands in 40% acetic acid according to standard procedure. For long term storage, place slides with squashed chromosomes in 100% 2-propanol at 20C. Prior to the in situ hybridization, denature the chromosomal D N A according to this procedure:
1 Rehydrate the slides by incubating them,
I. Labeling the hybridization probe

For best labeling results, follow the random primed D N A labeling procedure (Procedure IA) in C hapter 4 of this manual. We suggest a modification of this procedure as described below:
1 U se 0.51 g linearized D N A in 16 l
2 D enature in a boiling water bath for
redistilled H 2O .
3 Add 4 l D IG -H igh Prime reaction

4 Mix, centrifuge and incubate either 2 h at
10 min and chill on ice. mix.
for 2 min each, in solutions containing decreasing amounts of ethanol: 70% ethanol, 50% ethanol, 30% ethanol, 0.1 x SSC 2 x SSC . 2 D igest with RN ase if necessary. N ote: T his is usually not necessary.
3 For better preservation of the chromo-
37 C or overnight at room temperature. 5 Stop the labeling procedure by heating in boiling water for 10 min. 6 Add water and 20 x SSC to give a final volume of 100 l with a final concentration of 5 x SSC . N ote: I f a larger num ber of preparations is to be hybridiz ed, increase the v olum e to 200 l.
7 Add 1 volume of 10% SDS to 100 volumes
of the mixture in Step 6 (i.e., 1 l SDS to 100 l of mixture) to give a final concentration of 0.1% SDS. 8 The mixture is ready for hybridization and can be stored at 20C for at least several years without any significant decrease in hybridization efficiency. C omment: I n m y experience, it is absolutely unnecessary to rem ov e unincorporated dN T Ps from the m ixture before using the labeled probe. O n the contrary, purification steps lead to the loss of hybridiz able probe D N A and thus to a decrease in hybridiz ation efficiency.
somes, include a heat stabilization step by incubating slides in 2 x SSC for at least 30 min at 80C . 4 Incubate the slides for 90 s in 0.1 N N aO H , at room temperature. 5 Wash slides for 30 s in 2 x SSC . 6 D ehydrate slides by incubating them, for 2 min each, in solutions containing increasing amounts of ethanol: 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol. 7 Air dry the slides for 5 min. The preparations are then ready for hybridization. C omment: I f denatured chrom osom e preparations are to be stored for a long tim e, store them in 95% ethanol or 2-propanol at 20 C .
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Multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems

E. J. M . Speel, F. C . S. R am aek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands.
Ethanol suspension: Trypsinize cells (if
Fluorescence in situ hybridization (ISH ) is widely utilized because of its high sensitivity, resolution, and ability to detect multiple cellular nucleic acid sequences in different colors. Fluorescence ISH , however, has disadvantages, such as: Fluorescence signals fade when they are exposed to light. Autofluorescence in, e.g., tissue sections can interfere with target analysis. H ere we outline a multicolor enzyme-based cytochemical detection protocol for nucleic acids in situ . This protocol produces permanent cell preparations with non-diffusible, nonfading reaction products. The reaction products can be analyzed with brightfield (Speel et al., 1994a), reflection-contrast (Speel et al., 1993), or, in one case (alkaline phosphatase-Fast Red reaction), fluorescence microscopy (Speel et al., 1992). We show examples of single- and multipletarget ISH experiments on standard human lymphocyte metaphase spreads, as well as on interphase cell preparations. These results demonstrate the potential of this detection methodology for metaphase and interphase cytogenetics, e.g., for studying chromosome aberrations in different cell types (Martini et al., 1995). In addition, this methodology can be applied in the area of pathology, since the universal detection protocol described here can be combined with other sample preparation procedures, e.g., for tissue sections (H opman et al., 1991, 1992). The procedures given below are modifications of previously published procedures (Speel et al., 1992, 1993, 1994a, 1994b).
necessary), harvest, wash in PBS, fix in cold 70% ethanol (20C ), and drop onto glass slides that have been coated with poly-L-lysine. Slide and coverslip preparations: G row cells on glass slides or coverslips. Fix in cold methanol (20 C ) for 5 s, then in cold acetone (4 C ) for 3 x 5 s. Air dry samples and store at 20C . N ote: A lternativ ely, use other fixativ es for the slide and cov erslip preparations. C ytospins: C ytospin floating cells onto glass slides at 1000 rpm for 5 min. Air dry samples for 1 h at room temperature. Fix and store as with slide and coverslip preparations above.
II. Cell processing

1 D ecide whether samples need to be
I. Cell preparations
Lym phocytes: Prepare chromosomes from peripheral blood lymphocytes by standard methods. Fix in methanol:acetic acid (3:1, v/ v), and drop chromosomes onto glass slides that have been cleaned with a 1:1 mix of ethanol and ether. C ultured cells: Make preparations from cultured normal diploid cells or tumor cell lines by one of the following methods:
treated with RN ase. Then do one of the following: I f the cells need RN ase, go to Step 2. I f the cells do not need RN ase, go to Step 3. 2 Treat slides with RN ase as follows: O verlay each sample with 100 l RN ase solution (100 g/ ml RN ase A in 2 x SSC ) and a coverslip. Incubate cell samples for 1 h at 37 C . Remove coverslip and wash samples 3 x 5 min with 2 x SSC . G o to Step 3. 3 Treat slides with pepsin as follows: O verlay each sample with 100 l pepsin solution (50100 g/ ml pepsin in 10 mM H C l) and a coverslip. Incubate cell samples for 1020 min at 37C . Wash samples as follows: 2 min with 10 mM H C l 2 x 5 min with PBS 4 Post-fix samples as follows: Incubate samples with PBS containing 1% (para)formaldehyde for either 20 min at 4C or 10 min at room temperature. Wash slides 2 x 5 min with PBS. D ehydrate samples by passing slides through a series of ethanol solutions (70% , then 96% , then 100% ethanol), incubating 1060 s in each solution.
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III. Probe preparation

1 Label the D N A probes (containing either
IV . Multiple-target in situ hybridization (ISH)

IVA. ISHwith simultaneous probe and target denaturation [for probes with unique or highly repetitive (e.g., centromere probe) sequences]
1 O n each sample, place 10 l of hybridi-
repetitive or unique sequences) with Biotin-, D igoxigenin-, or FluoresceindU TP according to the nick translation procedure in C hapter 4 of this manual. 2 Just before use, prepare hybridization buffer containing: 50% or 60% formamide. 10% dextran sulfate. 2 x SSC . 0.2 g/ l sonicated herring sperm D N A. 0.2 g/ l yeast tRN A. 12 ng labeled probe D N A/ l hybridization buffer [if the probe contains unique or highly repetitive (e.g. centromere probe) sequences] or 24 ng labeled probe D N A/ l hybridization buffer, together with an excess (1001000 fold) of total human D N A or C o t-1 D N A [if the probe contains repetitive (e.g. A lu ) elements]. 3 Perform ISH by one of the following methods: I f the probe contains unique or highly repetitive (e.g. centromere probe) sequences, follow procedure IVA. I f the probe contains repetitive (e.g. A lu ) elements, follow procedure IVB. For multiple-target in situ hybridization, prepare D N A probes labeled with different haptens (biotin, digoxigenin, or fluorescein), mix them together, and follow either Procedure IVA or Procedure IVB (depending on the nature of the probes).
zation buffer containing labeled probe D N A (prepared as in Procedure III, Step 2). 2 C over sample with a 20 x 20 mm cover slip and (if you wish) seal the coverslip to the slide with rubber cement. 3 D enature probe and cellular D N A simultaneously by placing slides at 70 75C for 35 min on the bottom of a metal box. 4 Incubate hybridization samples over night at 37C . 5 G o to Procedure V.
IVB. ISH with separate probe and target denaturation [for probes with repetitive (e.g. Alu) elements]
1 Incubate the probe mixture (labeled
probe D N A, human or C o t-1 D N A, hybridization buffer; prepared as in Procedure III, Step 2) for 5 min at 75C . 2 C hill the denatured probe mixture on ice. 3 Incubate the denatured probe mixture for 14 h at 37C to pre-anneal the repetitive sequences in the mixture. 4 D enature the cell samples as follows: O verlay the cell samples with 70% formamide in 2 x SSC . Incubate the slides for 2 min at 70C to denature the cells. D ehydrate the cell samples in a series of chilled (20 C ) ethanol solutions (70% , then 96% , then 100% ethanol), incubating them for 5 min in each solution. Air dry the samples. 5 O n each denatured cell sample (from Step 4), place 10 l denatured, preannealed probe mixture (from Step 3). 6 Incubate hybridization samples over night at 37C . 7 G o to Procedure V.
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V . Post-hybridization washes
1 Perform the following stringent washes
6 Repeat Step 5 with the next incubation
of the samples (from either Procedure IVA or Procedure IVB) at 42C : 2 x 5 min with 2 x SSC containing 50% (or 60% ) formamide and 0.05% Tween 20. 2 x 5 min with 2 x SSC . 2 D epending upon the nature of the probe, do one of the following: I f the probe contains repetitive (e.g. A lu ) elements, wash the samples 2 x 5 min at 60C with 0.01 x SSC . I f the probe does not contain repetitive elements, skip this step.
VI. Enzyme-based cytochemical detection

VIA. Single color detection
Table 1: Frequently used detection Probe systems for enzyme-based in situ label hybridization. 1 Abbreviations used: Ab, antibody; ABC, avidin-biotinylated enzyme (horseradish peroxidase or alkaline phosphatase) complex; E, enzyme (horseradish peroxidase or alkaline phosphatase). 2 Hapten = biotin, digoxigenin, FITC, or DNP . 3 Anti-hapten Ab raised in another species (e.g., rabbit, goat, swine) can also be used as primary Ab in probe detection schemes.
in the detection system (Table 1) until all incubations are complete. 7 After all incubations in the detection system are complete, wash samples 5 min with PBS. 8 Visualize according to one of the proce dures in Section VII. 9 If the detection procedure uses the same enzyme (peroxidase or alkaline phosphatase) to detect two different probes, do the following: Inactivate the enzyme on the first detecting molecule by incubating the sample for 10 min at room temperature with 10 mM H C l. Repeat Steps 58 with a second detection system that recognizes the sec-ond probe (Speel et al., 1994b).
1 Avidin-E 1 Avidin-E Anti-hapten Ab-E Mouse3 anti-hapten Ab Mouse anti-hapten Ab Mouse anti-hapten Ab Mouse anti-hapten Ab
Incubation 2 Biotinylated anti-avidin Ab Anti-mouse Ab-E Rabbit anti-mouse Ab-E Biotinylated anti-mouse Ab D IG -labeled anti-mouse Ab
3 Avidin-E
Biotin Biotin H apten 2 H apten H apten H apten H apten
Anti-rabbit Ab-E ABC Anti-D IG Ab-E
1 Wash samples briefly with 4 x SSC
VIB. Multiple target, multicolor detection
containing 0.05% Tween 20. 2 Incubate samples for 10 min at 37 C with 4 x SSC containing 5% nonfat dry milk. 3 C hoose an appropriate enzyme-based detection system (Table 1). 4 D ilute detecting molecules as follows: D ilute avidin conjugates in 4 x SSC containing 5% nonfat dry milk. D ilute antibody conjugates in PBS containing 25% normal serum and 0.05% Tween 20. 5 For the first incubation in the detection system (Table 1), do the following: Incubate samples with diluted detecting molecule for 30 min at 37C . Wash samples 2 x 5 min in the appropriate wash buffer (4 x SSC for avidin; PBS for antibodies) containing 0.05% Tween 20.
To detect multiple probes labeled with different haptens, use a combination of detection systems. For example, Table 2 outlines a protocol for triple-target in situ hybridization. For protocols with doubletarget in situ hybridizations, see H opman et al. (1986), Emmerich et al. (1989), Mullink et al. (1989), H errington et al. (1989), Kerstens et al. (1994), and Speel et al. (1995). N ote: A lternativ ely, if tw o peroxidase or phosphatase reactions are used in the detection protocol (Speel et al., 1994b), inactiv ate the enz ym e after the first detection reaction in 10 m M H C l for 10 m in at room tem perature, then perform the second detection reaction (as in Procedure V I A abov e).
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D etection step 1. D etect biotin with AvPO 1 (diluted 1:50)
Incubation time2 20 min 5 min 10 min 30 min 30 min
Incubation temperature 37C 37 C RT 37C 37C
Table 2: Detection protocol for triple-target in situ hybridization

with a biotin-, digoxigenin-, and a FITC-labeled probe. 1 Abbreviations used: Ab, antibody; APase, alkaline phosphatase; AvPO, PO-conjugated avidin (Vector); DAB, diaminobenzidine; G AMAPase, APase-conjugated goat anti-mouse Ab (DAKO); MADIG , mouse anti-digoxigenin Ab; PO, horseradish peroxidase; RAFITC, rabbit anti-FITC Ab (DAKO); RT , room temperature; SW ARPO, PO-conjugated swine anti-rabbit Ab (DAKO). 2 For details of detection reactions, see Procedure VIA. For details of visualization reactions, see Procedures VIIAVIID. 3 For details of protein matrix, see Procedure VIII.
2. Visualize PO by Procedure VIIA (PO -D AB, brown signal) 3. Inactivate residual AvPO with 10 mM H C l. 4. D etect digoxigenin and FITC with MAD IG and RAFITC (each diluted 1:2000) 5. D etect anti-digoxigenin and anti-FITC with G AMAPase (diluted 1:25) and SWARPO (diluted 1:100) 6. Visualize APase activity by Procedure VIIC (APase-Fast Red, red signal) 7. Visualize PO activity by Procedure VIIB (PO -TMB, green signal) 8. C ounterstain with hematoxylin 9. Air dry 10. Embed in a protein matrix3
510 min 12 min 1 sec 10 min 10 min
37C 37 C RT RT 37C
VII. Visualization
Enzyme label PO 2 APase Substrate H 2O 2/ D AB H 2O 2/ TMB N -ASMX-P/ FR BC IP/ N BT Precipitate colors in microscopy Brightfield Reflection-contrast Fluorescence Brown G reen Red Purple White Pink/ Red Yellow Yellow/ O range Red
Table 3: Enzyme reaction
protocols and colors in different types of light microscopy1.
D epending upon the type of microscopy to be used for analysis (Table 3) and the detecting molecule, choose one of the following procedures to visualize the hybrids. In our hands, each of these procedures is optimal for in situ hybridization.
VIIA. Horseradish peroxidasediaminobenzidine (PO-DAB)
1 Mix color reagent just before use: 1 ml 3,3-diaminobenzidine tetra-
VIIB. Horseradish peroxidasetetramethylbenzidine (PO-TMB)

1 D issolve 100 mg sodium tungstate
chloride (D AB; Sigma) stock (5 mg D AB/ ml PBS). 9 ml PBS containing 0.1 M imidazole, pH 7.6. 10 l 30% H 2O 2. 2 O verlay each sample with 100 l color reagent and a coverslip. 3 Incubate samples for 515 min at 37C . 4 Wash samples 3 x 5 min with PBS and (if you wish) dehydrate them. 5 C overslip with an aqueous or organic mounting medium.
(Sigma) in 7.5 ml 100 mM citratephosphate buffer (pH 5.1). Adjust the pH of the tungstate solution to pH 5.05.5 with 37% H C l. 2 Just before use, dissolve 20 mg dioctyl sodium sulfosuccinate (Sigma) and 6 mg 3,3',5,5'-tetramethylbenzidine (TMB, Sigma) in 2.5 ml 100% ethanol at 80C . 3 Prepare 10 ml color reagent by combining the tungstate solution (Step 1), the TMB solution (Step 2), and 10 l 30% H 2O 2. 4 O verlay each sample with 100 l color reagent and a coverslip. 5 Incubate samples for 12 min at 37C . 6 Wash samples 3 x 1 min with ice-cold 100 mM phosphate buffer (pH 6.0) and (if you wish) dehydrate them. 7 C overslip with an organic mounting medium or immersion oil.
1 Other enzyme reactions that have been used for ISH are described in Speel et al. (1993, 1995). 2 Abbreviations used: APase, alkaline phosphatase; BCIP , bromochloro-indolyl phosphate; DAB, diaminobenzidine; FR, Fast Red TR; N-ASMX-P , naphthol-ASMXphosphate; NBT , nitroblue tetrazolium; PO, horseradish peroxidase; TMB, tetramethylbenzidine.
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VIIC. Alkaline phosphatase-Fast Red (APase-Fast Red)

1 Mix color reagent just before use: 4 ml TM buffer [200 mM Tris-H C l
Results
(pH 8.5), 10 mM MgC l2] containing 5% polyvinyl alcohol (PVA, MW 40,000; Sigma). 250 l TM buffer containing 1 mg naphthol-ASMX-phosphate (Sigma). 750 l TM buffer containing 5 mg Fast Red TR salt (Sigma). 2 O verlay each sample with 100 l color reagent and a coverslip. 3 Incubate samples for 515 min at 37C . 4 Wash samples 3 x 5 min with PBS. 5 C overslip with an aqueous mounting medium.
VIID. Alkaline phosphatase-bromochloroindolyl phosphate (APase-BCIP/NBT)
Follow the standard procedure given in C hapter 2 of this manual.
VIII. Embedding and light microscopy

1 Prepare samples for microscopy by
doing either of the following: I f samples require a single mounting medium, embed stained samples as described in Procedure VII. I f multiple precipitation reactions would require different (aqueous or organic) embedding mediums, apply instead a protein embedding layer by smearing 50 l of a 1:1 mixture of BSA solution (40 mg/ ml in deionized H 2O ) and 4% formaldehyde onto the slides. Air dry for 10 min at 37C . N ote: T his protein em bedding layer can be used to prev ent solubiliz ation of enz ym e precipitates during all types of light m icroscopy (Speel et al., 1993, 1994a). 2 Perform brightfield, fluorescence and reflection contrast microscopy as described in the literature (Speel et al., 1992, 1993, 1994b; Cornelese-Ten Velde et al., 1989).
Figure 1: Single-target ISH on a normal human lymphocyte metaphase spread with a peroxidase detection system. The probe was a biotinylated cosmid that recognizes 40 kb of chromosome 11q23. The detection system included monoclonal anti-biotin Ab (DAKO), rabbit anti-mouse Ab-PO, and the PO-DABreaction. The sample was counterstained with hematoxylin and viewed by brightfield microscopy.
Figure 2: Double-target ISH on normal human umbilical vein endothelial cells with brightfield viewing. The centromere of chromosome 1 (brown) was detected with a biotinylated probe; that of chromosome 7 (red), with a digoxigenin-labeled probe. The detection steps included (1) avidin-PO, (2) monoclonal anti-digoxigenin Ab and rabbit antimouse Ab-APase, (3) the APase-Fast Red reaction, and (4) the PO-DAB reaction. The sample was counterstained with hematoxylin and viewed by brightfield microscopy.
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Figure 3: Same ISH experiment as in Figure 1, but with a fluorescent alkaline phosphatase detection system. The detection system for the biotinylated cosmid probe included monoclonal anti-biotin, horse anti-mouse Ab-biotin, avidinbiotinylated APase complex, and the APase-Fast Red reaction (Speel et al., 1994a). The sample was counterstained with Thiazole Orange (Molecular Probes) and viewed by fluorescence microscopy.
Figure 4: Triple-target ISHon a normal human lymphocyte metaphase spread. Probes specific for the centromeres of chromosomes 1 (brown), 7 (red), and 17 (green) were labeled with biotin, digoxigenin, and FITC, respectively. Detection, counterstaining, and embedding in a BSA protein layer was as outlined in the text (Table 2). The sample was viewed by brightfield microscopy.
Figure 5: Triple-target ISH on human bladder tumor cell line T24. The probes and experimental procedures were the same as in Figure 4.
Reprinted from Speel, E. J. M.; Kamps, M.; Bonnet, J.; Ramaekers, F . C. S.; Hopman, A. H. N.; (1993) Multicolor preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy. Histochemistry 100, 357366 with permission of Springer-Verlag G mbH & Co. KG .
Figure 6: Double-target ISH on a normal human lymphocyte metaphase spread with reflection contrast viewing. The centromere of chromosome1 (yellow) was detected with a biotinylated probe; that of chromosome 17 (white), with a digoxigeninlabeled probe. The detection steps included (1) monoclonal anti-biotin Ab and rabbit anti-digoxigenin Ab, (2) horse anti-mouse Ab-biotin and swine anti-rabbit Ab-PO, (3) streptavidin-biotinylated APase complex, (4) the APase-Fast Red reaction, and (5) the PO-DAB reaction (Speel et al., 1993). The sample was not counterstained, but was embedded in a BSA protein layer and viewed by reflection contrast microscopy.
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References
Cornelese-Ten Velde, I.; Wiegant, J.; Tanke, H. J.; Ploem, J. S. (1989) Improved detection and quantification of the (immuno)peroxidase product using reflection contrast microscopy. Histochemistry 92, 153160. Emmerich, P .; Loos, P .; Jauch, A.; Hopman, A. H. N.; Wiegant, J.; Higgins, M. J.; White, B. N.; Van der Ploeg, M.; Cremer, C.; Cremer, T . (1989) Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography. Exp. Cell Res. 181, 126140. Herrington, C. S.; Burns, J.; G raham, A. K.; Bhatt, B.; McG ee, J. OD. (1989) Interphase cytogenetics using biotin and digoxygenin labeled probes II: simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei. J. Clin. Pathol. 42, 601606. Hopman, A. H. N.; Wiegant, J.; Raap, A. K.; Landegent, J. E.; Van der Ploeg, M.; Van Duijn, P . (1986) Bi-color detection of two target DNAs by non-radioactive in situ hybridization. Histochemistry 85, 14. Hopman, A. H. N.; Van Hooren, E.; Van de Kaa, C. A.; Vooijs, G .P .; Ramaekers, F . C. S. (1991) Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Modern Pathol. 4, 503513. Hopman, A. H. N.; Poddighe, P . J.; Moesker, O.; Ramaekers, F . C. S. (1992) Interphase cytogenetics: an approach to the detection of genetic aberrations in tumours. In: McG ee, J. OD.; Herrington, C. S. (Eds.) Diagnostic Molecular Pathology, A Practical Approach, 1st ed. New Y ork: IRL Press, 141167. Kerstens, H. M. J.; Poddighe, P . J.; Hanselaar, A. G . J. M. (1994) Double-target in situ hybridization in brightfield microscopy. J. Histochem. Cytochem. 42, 10711077. Martini. E.; Speel, E. J. M.; G eraedts, J. P . M.; Ramaekers, F . C. S.; Hopman, A. H. N. (1995) Application of different in situ hybridization detection methods for human sperm analysis. Hum. Reprod. 10, 855861. Mullink, H.; W alboomers, J. M. M.; Raap, A. K.; Meyer, C. J. L. M. (1989) Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes. Histochemistry 91, 195198. Speel, E. J. M.; Schutte, B.; Wiegant, J.; Ramaekers, F . C. S.; Hopman, A. H. N. (1992) A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-Fast Red reaction. J. Histochem. Cytochem. 40, 12991308. Speel, E. J. M.; Kamps, M.; Bonnet, J.; Ramaekers, F . C. S.; Hopman, A. H. N. (1993) Multicolour preparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflection contrast microscopy. Histochemistry 100, 357366. Speel, E. J. M.; Herbergs, J.; Ramaekers, F . C. S.; Hopman, A. H. N. (1994a) Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. J. Histochem. Cytochem. 42, 961966. Speel, E. J. M.; Jansen, M. P . H. M.; Ramaekers, F . C. S.; Hopman, A. H. N. (1994b) A novel triplecolor detection procedure for brightfield microscopy, combining in situ hybridization with immuocytochemistry. J. Histochem. Cytochem. 42, 12991307. Speel, E. J. M.; Ramaekers, F . C. S.; Hopman, A. H. N. (1995) Detection systems for in situ hybridization, and the combination with immunocytochemistry. Histochem. J., in press.
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Reagent RNase A Pepsin Digoxigenin-11-dUTP , alkali-stable Description Dry powder Aspartic endopeptidase with broad specificity Tetralithium salt, 1 mM solution Cat. No. 109 142 109 169 108 057 1 693 387 1 093 088 1 558 706 1 570 013 Fluorescein-12-dUTP Tetramethylrhodamine-6-dUTP AMCA-6-dUTP Biotin-16-dUTP DNase I DNA Polymerase I tRNA DNA, Cot-1, human Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Lyophilizate Nick Translation G rade From bakers yeast Solution in 10 mM Tris-HCl, 1 mM EDTA, pH 7.4, Cot-1 DNA is used in chromosomal in situ suppression (CISS). 1 373 242 1 534 378 1 534 386 1 093 070 104 159 104 485 104 493 109 495 109 509 1 581 074 Pack size 25 mg 100 mg 1g 5g 25 nmol (25 l) 125 nmol (125 l) 5x125 nmol (5x125 l) 25 nmol (25 l) 25 nmol (25 l) 25 nmol (25 l) 50 nmol (50 l) 100 mg 500 units 1000 units 100 mg 500 mg 500 g (500 l) 5x10 ml 100 g 200 g 150 U 150 U 8 ml
Tween 20 Anti-Digoxigenin Anti-Mouse IgDigoxigenin Anti-Digoxigenin-POD Anti-Digoxigenin-AP NBT/BCIP , (stock solution) Clone 1.71.256, mouse IgG 1, F(ab)2 fragment Fab fragments from sheep Fab fragments from sheep Solution of 18.75 mg/ml nitro-blue tetrazolium chloride and 9.4 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, toluidine-salt in 67% DMSO (v/v) Powder
1 332 465 1 333 062 1 214 624 1 207 733 1 093 274 1 681 451
Tris
127 434 708 968 708 976 238 031 238 040
100 g 500 g 1 kg 1g 10 g
BSA
Highest quality, lyophilizate
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107
DNA in situ hybridization with an alkaline phosphatase-based fluorescent detection system

D r. G . Sagner, R esearch L aboratories, Boehringer M annheim G m bH , Penz berg, G erm any.
Prepare Fast Red TR stock solution
We describe here a high sensitivity indirect detection procedure for D IG -labeled hybridization probes. The procedure uses the components of the H N PP Fluorescent Detection Set to form a fluorescent precipitate of H N PP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) and Fast Red TR at the site of hybridization. This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes.
I. In situ hybridization with DIG -labeled probes

Prepare chromosomal spreads, label hybridization probes with digoxigenin (D IG ), hybridize probes to chromosomes, and perform posthybridization washes of the sample according to standard procedures, e.g. those listed in C hapters 4 and 5 of this manual.
by dissolving 5 mg Fast Red TR (vial 2) in 200 l distilled H 2O . Mix 10 l H N PP stock solution (premixed, vial 1), 10 ml Fast Red TR stock solution, and 1 ml detection buffer [100 mM Tris-H C l, 100 mM N aC l, 10 mM MgC l2; pH 8.0 (20 C )]. Filter the H N PP/ Fast Red TR mix through a 0.2 m nylon filter. C aution: T he Fast R ed T R stock solution should be no m ore than 4 w eek s old. T he H N PP/ Fast R ed T R m ix should be no m ore than a few days old. A lw ays filter the m ix just before use.
6 C over each sample with 100 l filtered
II. Detection of DIG -labeled probes

1 Prepare a fresh 1:500 dilution of alkaline
phosphatase-conjugated sheep anti-D IG antibody in blocking buffer [100 mM Tris-H C l, 150 mM N aC l, 0.5% blocking reagent; pH 7.5 (20C )]. N ote: D o not store the diluted antibody conjugate m ore than 12 h at 4 C .
2 Pipette 100 l of diluted anti-D IG
H N PP/ Fast Red TR mix and a coverslip, then incubate slides for 30 min at room temperature. 7 Wash the slides 1x at room temperature with washing buffer [100 mM Tris-H C l, 150 mM N aC l, 0.05% Tween 20; pH 7.5 (20 C )]. 8 Repeat Steps 6 and 7 three times. N ote: I f you are detecting abundant or m ultiple copy sequences, sk ip Step 8. O ne incubation is enough.
9 Wash the slides for 10 min with distilled
H 2O .
III. Fluorescence microscopy

1 In a C oplin jar, counterstain slides with
antibody conjugate onto each slide and add a coverslip. 3 Incubate slides for 1 h at 37C in a moist chamber. 4 Wash the slides at room temperature as follows: 3 x 10 min with washing buffer [100 mM Tris-H C l, 150 mM N aC l, 0.05% Tween 20; pH 7.5 (20 C )]. 2 x 10 min with detection buffer [100 mM Tris-H C l,100 mM N aC l, 10 mM MgC l2; pH 8.0 (20 C )]. 5 Prepare fresh H N PP/ Fast Red TR mix as follows: N ote: N um bered v ials are com ponents of the H N PP Fluorescent D etection Set.
50 ml D API solution (100 ng D API/ ml PBS) for 5 min in the dark at room temperature. 2 Wash slides under running water for 23 min. 3 Air dry slides in the dark. 4 Add 20 l D ABC O antifading solution (PBS containing 50% glycerol and 2% D ABC O ) to each slide. 5 C over each slide with a 24 x 24 mm coverslip. N ote: For long term storage of the slides, seal the edges of the cov erslip w ith nail polish and store in the dark at 20 C .
6 View the slides by fluorescence micros
copy, using appropriate filters.
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N ote: T he m axim um em ission w av elength of dephosphorylated H N PP/ Fast R ed T R is 562 nm . H ow ev er, since the em ission peak is broad (540590 nm ), you m ay use the filter sets for either fluorescein or rhodam ine to analyz e the H N PP/ Fast R ed T R product.
Results
The fluorescent signal obtained with the H N PP/ Fast Red procedure described above is more intense than a direct signal from fluorescein. In a comparison experiment (Figure 1), the H N PP/ Fast Red reaction product was visible after a 70-fold shorter exposure (0.2 sec exposure vs. 15 s exposure) than the fluorescein signal. Even when the H N PP/ Fast Red signal is viewed under a less than optimal filter (Figure 2), the fluorescence is clearly visible.
Figure 1: Comparison of fluorescein and HNPP signal intensity. A DIG-labeled painting
probe specific for human chromosome 1 was hybridized to metaphase chromosomal spreads. The DIG -labeled probe was detected with (Panel a) anti-DIG -fluorescein conjugate or (Panel b) anti-DIG -alkaline phosphate conjugate and the HNPP/Fast Red detection reaction described in the text. The fluorescein signal (Panel a) required a 15 s exposure while the HNPP/Fast Red signal (Panel b) required a 0.2 s exposure. Fluorescent images were pseudocolored with a CCD camera and a digital imaging system. Chromosomes were counterstained with DAPI.
Figure 2: HNPP/Fast Red

Reagent Tris Description Powder Cat. No. 127 434 708 968 708 976 1 096 176 1 093 274 1 332 465 For sensitive fluorescent detection of nonradioactively labeled nucleic acids in fluorescence in situ hybridization (FISH) and membrane hybridization Fluorescence dye for staining of chromosomes 1758 888 Pack size 100 g 500 g 1 kg 50 g 150 U 5x10 ml Set for 500 FISH reactions
fluorescence viewed with a filter for DAPI. A DIG -labeled painting probe specific for human chromosome 1 was hybridized to metaphase chromosomal spreads, then detected with anti-DIG -alkaline phosphate conjugate and the HNPP/Fast Red detection reaction as in Figure 1. The HNPP/Fast Red signal was viewed under a filter that was optimal for DAPI (emission maximum, 470 nm), but not for HNPP (emission maximum, 562 nm). The fluorescence was photographed directly without digital manipulation. Chromosomes were counterstained with DAPI.
Blocking Reagent Anti-Digoxigenin-AP Tween 20 HNPP Fluorescent Detection Set
Powder Fab Fragments from sheep
DAPI
236 276
10 mg
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109
Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to cells
Combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations
E. J. M . Speel, F. C . S. R am aek ers, and A . H . N . H opm an, D epartm ent of M olecular C ell Biology & G enetics, U niv ersity of L im burg, M aastricht, T he N etherlands.
The combination of in situ hybridization (ISH ) and immunocytochemistry (IC C ) enables us, e.g., to simultaneously demonstrate mRN A and its protein product in the same cell, to immunophenotype cells containing a specific chromosomal aberration or viral infection, or to characterize cytokinetic parameters of tumor cell populations that are genetically or phenotypically aberrant. The factors that determine the success and sensitivity of a combination IC C and nonradioactive ISH procedure include:
Preservation of cell morphology and
Fluorochrom es have been used mainly on acetone-fixed cell preparations, since the material can be mildly post-fixed (usually with paraformaldehyde) after antigen detection and used directly for fluorescence ISH without any further pretreatment (Weber-Matthiesen et al., 1993). H owever, amplification steps for both IC C and ISH signals are often necessary for clear visualization. In such cases, enzymatic ISH pre-treatment after IC C lowers fluorescent IC C staining dramatically (Speel et al., 1994a). Enz ym e precipitation reactions have also been used efficiently for combined IC C / ISH staining of proteins in cell preparations and tissue sections. Enzyme precipitation products that withstand the proteolytic digestion and denaturation steps used in the ISH procedure include:
Several precipitates (Fast Red, N ew
protein epitopes Accessibility of nucleic acid targets Lack of cross-reaction between the different detection procedures G ood color contrast Stability of enzyme cytochemical precipitates and fluorochromes Since several steps in the ISH procedure (enzymatic digestion, post-fixation, denaturation at high temperatures, and hybridization in formamide) may destroy antigenic determinants, IC C usually precedes ISH in a combination procedure. A variety of such combined IC C / ISH procedures have been reported with either enzyme precipitation reactions (Mullink et al., 1989; Van den Brink et al., 1990; Knuutila et al., 1994; Speel et al., 1994b), fluorochromes (Van den Berg et al., 1991; Weber-Matthiesen et al., 1993), or a combination of both (Strehl & Ambros, 1993; Zheng et al., 1993; H erbergs et al., 1994; Speel et al., 1994a). The procedures can be subdivided into two groups, those which use fluorochromes for ICC, and those which use enzyme reactions.
Fuchsin, and BC IP/ N BT) formed by alkaline phosphatase The diaminobenzidine precipitate formed by horseradish peroxidase The BC IG (X-G al) precipitate formed by -galactosidase In these cases, the digestion and denaturation steps of the ISH procedure remove the antibody and enzyme detection layers, but the precipitate remains firmly in place. The stability of the IC C precipitate thus prevents unwanted cross-reaction between the detection procedures for ISH and IC C . H ere we present a combined IC C / ISH procedure which describes compatible detection systems for fluorescence or brightfield microscopy (Table 1). Additionally, this procedure allows the localization of incorporated BrdU by fluorescence microscopy.
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The procedures given below are modifications of previously published procedures (Speel et al., 1994a, 1994b).
Table 1: Detection systems for combined ICC/ISH1.
For analysis by Fluorescence microscopy Brightfield microscopy ICC 2 D etection by Anti-antigen Ab-APase Anti-antigen- -gal -G al-BC IG D IG - or biotin-labeled nucleic acid PO - or APase-labeled antiD IG Ab or anti-biotin Ab plus PO -D AB, PO -TMB, or APase-Fast Red reaction
Visualization by APase-Fast Red ISH Probe D etection and visualization by FITC - or AMC A-labeled nucleic acid D irect viewing
BrdU labeling
D etection by
AMC A- or FITC labeled 3 anti-BrdU Ab
Visualization by D irect viewing
1 Amplification steps may be necessary for detection of low amounts of antigen, nucleic acid target, or incorporated BrdU. 2 Abbreviations used: Ab, antibody; AMCA, aminomethylcoumarin acetic acid; APase, alkaline phosphatase; BCIG , bromochloroindolyl-galactoside; BrdU, bromodeoxyuridine; DAB, diaminobenzidine; DIG , digoxigenin; FITC, fluorescein isothiocyanate; -G al, galactosidase; ICC, immunocytochemistry; ISH, in situ hybridization; PO, peroxidase; TMB, tetramethylbenzidine 3 The fluorochrome used in the BrdU visualization step should be different from the one used in the ISH visualization step.
I. Cell preparations and BrdU labeling

1 (O ptional) To label cells, add BrdU
4 Incubate slides for 45 min at room
(final concentration, 10 M) to the culture medium 30 min before harvesting the cells (Speel et al., 1994a). 2 Prepare cultured normal diploid cells or tumor cell lines (labeled or unlabeled) by one of the following methods: Slide and coverslip preparations: Grow cells on glass slides or coverslips. Fix in cold methanol (20C) for 5 s, then in cold acetone (4C) for 3 x 5 s. Air dry samples and store at 20C. N ote: A lternativ ely, use other fixativ es for the slide and cov erslip preparations.
C ytospins: C ytospin floating cells
onto glass slides at 1000 rpm for 5 min. Air dry samples for 1 h at room temperature. Fix and store as with slide and coverslip preparations above.
temperature with an appropriate secondary antibody conjugate. U se the following to decide which antibody conjugate to use: I f you wish to detect the IC C antigen, the ISH antigen, and (optionally) BrdU labeling by fluorescence microscopy, use a secondary antibody that is conjugated to alkaline phosphatase (APase). I f you wish to detect the IC C antigen and the ISH antigen under brightfield microscopy, use a secondary antibody that is conjugated to -galactosidase (-G al). 5 Wash slides as follows: 5 min with PBS containing 0.05% Tween 20. 5 min with PBS. N ote: For am plification of the I C C signal, you m ay add a third antibody step after this w ash. For details of possible antibodies to use in an am plified three-antibody detection procedure, see Table 1 of the article M ultiple target D N A in situ hybridiz ation w ith enz ym e-based cytochem ical detection system s on page 102 of this m anual.
6 Visualize the antibody-antigen com
II. Detection of antigen by immunocytochemistry (ICC)

1 Incubate slides for 10 min at room
temperature with PBS-Tween-N GS (PBS buffer containing 0.05% Tween 20 and 25% normal goat serum). 2 Incubate slides for 45 min at room tem perature with an appropriate dilution of antigen-specific primary antibody in PBS-Tween-N G S. 3 Wash slides for 2 x 5 min with PBS containing 0.05% Tween 20.
plexes according to either Procedure IIIA (for APase conjugates) or Procedure IIIB (for -G al conjugates).
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III. Visualization of ICC antigen

IIIA. APase-Fast Red reaction (for producing a red precipitate visible under either fluorescence or brightfield microscopy)
1 Mix color reagent just before use: 4 ml TM buffer [200 mM Tris-H C l
IV. Cell processing for in situ hybridization

1 Wash slides for 2 min at 37C with
2 D igest samples with pepsin as follows: O verlay each sample with pepsin
10 mM H C l.
(pH 8.5), 10 mM MgC l2] containing 5% polyvinyl alcohol (PVA, MW 40,000; Sigma). 250 l TM buffer containing 1 mg naphthol-ASMX-phosphate (Sigma). 750 l TM buffer containing 5 mg Fast Red TR salt (Sigma). 2 O verlay each sample with 100 l color reagent and a coverslip. 3 Incubate samples for 515 min at 37C . C aution: M onitor the enz ym e reaction under a m icroscope and adjust the reaction tim e to k eep the precipitate from becom ing so dense that it shields nucleic acid sequences from the I SH detection step.
4 Wash samples 3 x 5 min with PBS.
solution (100 g/ ml pepsin in 10 mM H C l). Incubate cell samples for 1020 min at 37C . 3 Wash samples for 2 min at 37C with 10 mM H C l. N ote: For cells stained w ith the -G alBC I G reaction, you m ay (if you w ish) dehydrate the slides after w ashing them .
4 Post-fix samples for 20 min at 4C with 5 Wash samples as follows: 5 min with PBS. 5 min with 2 x SSC .
PBS containing 1% paraformaldehyde.
V . In situ hybridization (ISH)

1 Perform a standard ISH detection and
N ote: D o not dehydrate sam ples after w ashing.

IIIB. -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy)
1 Mix color reagent: 2.5 l 5-bromo-4-chloro-3-indolyl-
-D -galactoside (BC IG ; X-G al) stock solution [20 mg/ ml BC IG (X-G al) in N ,N -dimethylformamide]. 100 l diluent (PBS containing 0.9 mM MgC l2, 3 mM potassium ferricyanide, and 3 mM potassium ferrocyanide). 2 O verlay each sample with 100 l color reagent and a coverslip. 3 Incubate samples for 1560 min at 37C . C aution: M onitor the enz ym e reaction under a m icroscope and adjust the reaction tim e to k eep the precipitate from becom ing so dense that it shields nucleic acid sequences from the I SH detection step.
4 Wash samples 3 x 5 min with PBS.
visualization procedure on the slides as described elsewhere in this manual. U se either: Fluorescence-based procedures (FITC, AMC A) (when APase-conjugated antibodies were used for IC C ). Enzyme-based procedures (PO -D AB, PO -TMB, APase-Fast Red) (when -G al-conjugated antibodies were used for IC C ). Example: For a detailed description of enz ym e-based I SH procedures, see M ultiple target D N A in situ hybridiz ation w ith enz ym e-based cytochem ical detection system s on page 100 of this m anual.
2 Include appropriate controls in the ISH
procedure to ensure the lack of crossreaction between IC C and ISH . N ote: T he I C C precipitate rem ains firm ly in place during I SH . T he stability of the I C C precipitate usually prev ents unw anted cross-reaction betw een the detection procedures for I SH and I C C .
N ote: I f you w ish, dehydrate the sam ples after w ashing.
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VI. Fluorescence detection of BrdU (optional)

1 After performing the APase-Fast Red
IC C (Procedure IIIA) and fluorescence ISH (Procedure V) reactions on BrdU labeled cells, incubate the cells for 45 min at room temperature with a specific antiBrdU antibody. 2 Wash slides for 2 x 5 min with PBS containing 0.05% Tween 20. 3 Incubate samples with a secondary antibody that is conjugated to a fluorochrome not used in the IC C or ISH reactions. Example: U se an alk aline-phosphatase conjugated antibody and the A PaseFast R ed for I C C ; a FI T C -conjugated antibody for I SH ; and an A M C A conjugated antibody for BrdU detection. N ote: I f necessary, use an am plified three-antibody detection procedure as outlined in the article M ultiple target D N A in situ hybridiz ation w ith enz ym ebased cytochem ical detection system s on page 100 of this m anual.
4 Before embedding, wash slides as follows: 2 x 5 min with PBS containing 0.05%
For brightfield microscopy (for detection of -Gal-BCIG ICC and enzyme-based ISH ): D epending on the enzyme reactions used, embed specimens in one of the following: Aqueous, organic, or protein embedding medium, as described in the article Multiple target D N A in situ hybridization with enzyme-based cytochemical detection systems on page 100 of this manual. Entellan (Merck) organic embedding medium and immersion oil (Zeiss) (for peroxidase-D AB or peroxidase-TMB reactions only). Tris-glycerol mixture [1:9 (v/ v) mix of 200 mM Tris-H C l (pH 7.6) and glycerol] (for peroxidase-D AB or phosphataseFast Red reactions only).
Results
Figure 1: Combined ICC and fluorescence
ISH on lung tumor cell line EPLC65, showing cytokeratin filaments, chromosome 1, and chromosome 17. The cytokeratins (red) were visualized with a monoclonal anti-cytokeratin antibody (Ab), an alkaline phosphatase-conjugated goat anti-mouse Ab, and the APase-Fast Red reaction. The centromere of chromosome 1 (blue) was visualized with a biotinylated probe, avidinAMCA, a biotinylated goat anti-avidin Ab, and avidin-AMCA. The centromere of chromosome 17 (green) was visualized directly with a FITC-labeled probe.
5 Include appropriate controls to ensure
5 min with PBS.
Tween 20.
that the BrdU detection step does not cross-react with ISH detection.
VII. Embedding
For fluorescence microscopy (for detection of APase-Fast Red ICC, fluorescence ISH , and BrdU ): Embed specimens in a Tris-glycerol mixture [1:9 (v/ v) mix of 0.2 M Tris-H C l (pH 7.6) and glycerol] containing 2% D ABC O (Sigma) and (optionally) 0.5 g/ ml blue D API (Sigma).
Figure 2: Combined ICC and fluo-
rescence ISH on EPLC 65 cells, showing the nuclear proliferation marker Ki67, chromosome 7, and incorporated BrdU. Panel a: The Ki67 antigen (red) was visualized with a rabbit anti-Ki67 Ab, alkaline phosphatase-conjugated swine antirabbit Ab, and the APase-Fast Red reaction. The centromere of chromosome 7 (green) was visualized directly with a FITClabeled probe. Panel b: Incorporated BrdU (blue) was visualized with monoclonal anti-BrdU Ab, biotinylated horse anti-mouse Ab, and avidin-AMCA.
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Figure 3: Combined ICC and enzyme-based ISH on a human umbilical vein endothelial cell, showing the intermediate filament protein vimentin, chromosome 1, and chromosome 7. Vimentin (blue) was visualized with monoclonal anti-vimentin Ab, -galactosidase-conjugated goat anti-mouse Ab, and the -galactosidase-BCIG reaction. The centromeres of chromosome 1 (biotinylated probe, brown) and chromosome 7 (digoxigenin-labeled probe, red) were visualized with avidin-peroxidase, rabbit antidigoxigenin Ab and alkaline phosphatase-conjugated swine anti-rabbit Ab, the APase-Fast Red reaction, and the PO-DAB reaction. Samples were embedded in a Tris-glycerol mixture, but were not counterstained. Slides were viewed by brightfield microscopy.
References
Herbergs, J.; De Bruine, A. P .; Marx, P .T . J.; Vallinga, M. I. J.; Stockbrgger, R. W .; Ramaekers, F . C. S.; Arends, J. W .; Hopman, A. H. N. (1994) Chromosome aberrations in adenomas of the colon. Proof of trisomy 7 in tumor cells by combined interphase cytogenetics and immunocytochemistry. Int. J. Cancer 57, 781785. Strehl, S.; Ambros, P .F . (1993) Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma. Cytogenet. Cell G enet. 63, 2428. Van Den Berg, H.; Vossen, J. M.; Van Den Bergh, R. L.; Bayer, J.; Van Tol, M. J. D. (1991) Detection of Y chromosome by in situ hybridization in combination with membrane antigens by two-color immunofluorescence. Lab. Invest. 64, 623628. Van Den Brink, W .; Van Der Loos, C.; Volkers, H.; Lauwen, R.; Van Den Berg, F .; Houthoff, H.; Das, P . K. (1990) C ombined -galactosidase and immunogold/ silver staining for immunohistochemistry and DNA in situ hybridization. J. Histochem. C ytochem. 38, 325329. W eber-Matthiesen, K.; Deerberg, J.; Mller-Hermelink, A.; Schlegelberger, B.; G rote, W . (1993) Rapid immunophenotypic characterization of chromosomally aberrant cells by the new fiction enet. 63, 123125. method. Cytogenet. Cell. G Zheng, Y .-L.; Carter, N. P .; Price, C. M.; Colman, S. M.; Milton, P . J.; Hackett, G . A.; G reaves, M. F .; FergusonSmith, M. A. (1993) Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative enet. 30, 10511056. magnetic cell sorting. J. Med. G
Knuutila, S.; Nylund, S. J.; W essman, M.; Larramendy, M. L. (1994) Analysis of genotype and phenotype on the same interphase or mitotic cell. A manual of MAC (morphology antibody chromoenet. Cytogenet. 72, somes) methodology. Cancer G 115. Mullink, H.; W alboomers, J. M. M.; Tadema, T . M.; Jansen, D. J.; Meijer, C. J. L. M. (1989) Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences. J. Histochem. Cytochem. 37, 603609. Speel, E. J. M.; Herbergs, J.; Ramaekers, F . C. S.; Hopman, A. H. N. (1994a) Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. J. Histochem. Cytochem. 42, 961966. Speel, E. J. M.; Jansen, M. P . H. M.; Ramaekers, F . C. S.; Hopman, A. H. N. (1994b) Anovel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. J. Histochem. Cytochem. 42, 12991307. Speel, E. J. M.; Ramaekers, F . C. S.; Hopman, A. H. N. (1995) Detection systems for in situ hybridization, and the combination with immunocytochemistry. Histochem. J., in press.
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Reagent 5-Bromo-2'-deoxyuridine Pepsin Digoxigenin-11-dUTP , alkali-stable Description One tablet contains approx. 50 mg 5-bromo-2'-deoxy-uridine and approx. 20 mg binder. Aspartic endopeptidase with broad specificity Tetralithium salt, 1 mM solution Cat. No. 586 064 Pack size 50 tablets
108 057 1 693 387 1 093 088 1 558 706 1 570 013
1g 5g 25 nmol (25 l) 125 nmol (125 l) 5x125 nmol (5x125 l) 25 nmol (25 l) 25 nmol (25 l) 25 nmol (25 l) 50 nmol (50 l) 100 mg 500 units 1000 units 150 U (200 l)
Fluorescein-12-dUTP Tetramethylrhodamine-6-dUTP AMCA-6-dUTP Biotin-16-dUTP DNase I DNA Polymerase I Anti-Digoxigenin-AP
Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Lyophilizate Nick Translation G rade Fab Fragments from sheep
1 373 242 1 534 378 1 534 386 1 093 070 104 159 104 485 104 493 1 093 274
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115
In situ hybridization to mRNA in in vitro cultured cells with DNA probes

D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands. The protocol given below has been developed with a cell line (rat 9G ) which has integrated into its genome the major immediate early transcription unit 1 of human cytomegalovirus (Boom et al., 1986). The protocol is written in general terms and is applicable to abundantly expressed mRN A species. More information concerning this specific in situ hybridization application can be found in Raap et al. (1991).
II. In situ hybridization

1 Label a suitable probe D N A with dig-
I. Cell preparation, fixation and permeabilization

N ote: A ll solutions m ust be treated w ith R N ase inhibitors.
1 C ulture cells at 37 C in a 5% C O 2
atmosphere on poly-L-lysine coated microscopic slides. As culture medium, use Dulbeccos minimal essential medium without phenol red. 2 Wash the cells with PBS at 37C , then fix at room temperature for 30 min in a solution of 4% (w/ v) formaldehyde, 5% (v/ v) acetic acid, and 0.9% (w/ v) N aC l. 3 Wash the fixed cells with PBS at room temperature and store them in 70% ethanol at 4C . 4 Before in situ hybridization, treat the fixed cells as follows: D ehydrate by incubating successively in 70% , 90% , and 100% ethanol. Wash in 100% xylene to remove residual lipids. Rehydrate by incubating successively in 100% , 90% , and 70% ethanol. Finally, incubate in PBS. 5 Treat the fixed cells at 37C with 0.1% (w/v) pepsin in 0.1 N H Cl, to increase permeability to macromolecular reagents. 6 Finally, treat the fixed cells as follows: Wash with PBS for 5 min. Post-fix with 1% formaldehyde for 10 min. Wash again with PBS.
oxigenin (D IG ), using any of the protocols given elsewhere in this manual. 2 Prepare hybridization solution which contains 60% deionized formamide, 300 mM N aC l, 30 mM sodium citrate, 10 mM ED TA, 25 mM N aH 2PO 4 (pH 7.4), 5% dextran sulfate, and 250 ng/ l sheared salmon sperm D N A. 3 D enature the D IG -labeled probe D N A at 80 C shortly before use and add it to the hybridization solution at a concentration of 5 ng/ l. 4 Add 10 l of the hybridization mixture (hybridization solution plus denatured probe) to the fixed, permeabilized cells and cover with an 18 x 18 mm coverslip. N ote: A n in situ denaturation step is optional. I nclusion of such a step m ay intensify the R N A signal since it m ak es the sam ple m ore accessible to the probe.
5 H ybridize at 37C for 16 h.
III. W ashes
1 After hybridization, remove coverslips
by shaking the slides at room temperature in a solution of 60% formamide, 300 mM N aC l, and 30 mM sodium citrate . 2 U sing the same formamide-salt solution as in Step 1, wash the slides as follows: Wash 3 x at room temperature. Wash 1x at 37C . 3 Finally, wash the slides 1 x 5 min in PBS.
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IV . Immunofluorescent detection
N ote: O ther protocols for am plifying the signal m ay also be used. See the protocols under Single color fluorescent detection w ith im m unological am plification on page 62.
1 Block
7 Embed the cell samples in an anti-fading
non-specific binding by the following steps: Add 100 l blocking solution [100 mM Tris-H C l; pH 7.5, 150 mM N aC l, 0.5% (w/ v) blocking reagent] to each slide. C over with a 24 x 50 mm coverslip. Place slide in a moist chamber. 2 To loosen the coverslips, wash the slides briefly with the buffer used for immunological detection. 3 D etect the hybridized D IG -labeled probe by incubating the slides in a moist chamber for 45 min with a 1:500 dilution of anti-D IG -fluorescein in blocking solution (from Step 1) . 4 Wash slides with a solution of 100 mM Tris-H C l, pH 7.5; 150 mM N aC l; 0.05% Tween 20. 5 To dehydrate the cell samples, incubate the slides for 5 min in each of a series of ethanol solutions: 70% , 90% , then 100% ethanol. 6 Air dry the slides.
Figure 1: Nuclear staining of integrated IE viral DNA after induction with cycloheximide. The signal throughout the cytoplasm represents viral mRNA. The two panels show the same signal with different counterstains.
solution which contains: 9 parts glycerol plus 1 part staining mixture: 1 M TrisH C l (pH 7.5), 2% 1,4-diaza-bicyclo[2,2,2]-octane (D ABC O ), and a D N A counterstain [either propidium iodide (500 ng/ ml) or D API (75 ng/ l)].
Results
Panel a: PI counterstain.
5
Panel b: DAPI counterstain.
References
Boom, R.; G eelen, J. L.; Sol, C. J.; Raap, A. K.; Minnaar, R. P .; Klaver, B. P .; Noordaa, J. v. d. (1986) Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate early gene products by protein synthesis inhibition. J. Virol. 58, 851859. Raap, A. K.; V an de Rijke, F .M.; Dirks, R. W .; Sol, C. J.; Boom, R.; Van der Ploeg, M. (1991) Bicolor fluorescence in situ hybridization to intron- and exon mRNA sequences. Exp. Cell Res. 197, 319322.
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Reagent Pepsin Tween 20 Blocking Reagent Anti-Digoxigenin Fluorescein DAPI Digoxigenin-11-dUTP , alkali-stable Powder Fab Fragments from sheep Fluorescence dye for staining of chromosomes Tetralithium salt, 1 mM solution Description Aspartic endopeptidase with broad specificity Cat. No. 108 057 1 693 387 1 332 465 1 096 176 1 207 741 236 276 1 093 088 1 558 706 1 570 013 Fluorescein-12-dUTP Tetramethylrhodamine-6-dUTP AMCA-6-dUTP Biotin-16-dUTP DNase I DNA Polymerase I Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Tetralithium salt, 1 mM solution Lyophilizate Nick Translation G rade 1 373 242 1 534 378 1 534 386 1 093 070 104 159 104 485 104 493 808 261 808 270 808 288 Pack size 1g 5g 5x10 ml 50 g 200 g 10 mg 25 nmol (25 l) 125 nmol (125 l) 5x125 nmol (5x125 l) 25 nmol (25 l) 25 nmol (25 l) 25 nmol (25 l) 50 nmol (50 l) 100 mg 500 units 1000 units 250 g 500 g 1 kg
EDTA
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Identification of single bacterial cells using DIG -labeled oligonucleotides

B. Z arda, D r. R . A m ann, and Prof. D r. K.-H . Schleifer, I nstitute for M icrobiology, Technical U niv ersity of M unich, G erm any. N ote: A n earlier v ersion of this procedure has been published (Z arda et al., 1991).
I. O rganisms and growth conditions

1 To prepare cells needed for this experi-
II. Cell fixation and preparation of cell smears

N ote: T his procedure w as adapted from A m ann et al., 1990.
1 Fix the cells by adding 3 volumes of para-
ment, do the following: Allow Escherichia coli (D eutsche Sammlung von Mikroorganismen und Zellkulturen G mbH , D SM 30083) to grow aerobically in YT broth (tryptone, 10 g/L; yeast extract, 5 g/L; glucose, 5 g/L; sodium chloride, 8 g/L; pH 7.2) at 37C. C ultivate Pseudom onas cepacia (D SM 50181) aerobically in M1 broth (peptone, 5 g/ L; malt extract, 3 g/ L; pH 7.0) at 30C . Note: Cells from Methanococcus vannielii (D SM 1224) w ere a generous gift of D r. R . H uber (D ept. of M icrobiology, U niv ersity of R egensburg, FR G ).
2 To guarantee a high cellular rRN A
content, harvest all cells at midlogarithmic phase by centrifugation in a microcentrifuge (5000 x g, 1 min). 3 D iscard the growth medium from the cell pellet and resuspend cells thoroughly in phosphate buffered saline (130 mM sodium chloride, 10 mM sodium phosphate; pH 7.2).
formaldehyde solution (4% paraformaldehyde in PBS) to 1volume of suspended cells. 2 After 3 h incubation, do the following: Pellet cells by centrifugation. Remove the supernatant. Wash the cell pellet with PBS. Resuspending the cells in an aliquot of PBS. N ote: A fter adding 1 v olum e ethanol to the resuspended cells, you m ay store the cell suspension at 20C for up to 3 m onths w ithout apparent influence on the hybridiz ation results.
3 Spot these fixed cell suspensions onto
thoroughly cleaned glass slides and allow to air dry for at least 2 h. 4 D ehydrate the cell samples by immers ing the slides successively in solutions of 50% ethanol, then 80% ethanol, then 98% ethanol (3 min for each solution).
III. DIGlabeling of oligonucleotides with DIG -ddUTP

Label oligonucleotides either according to the protocols in C hapter 4 of this manual or at the 5' end according to the protocol of the D IG O ligonucleotide 5'-End Labeling Set*.
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119
IV . In situ hybridization using digoxigenin-labeled oligonucleotides

1 Prepare hybridization solution (900 mM
5 View the cell smears with a Plan
sodium chloride, 20 mM Tris-H C l, 0.01% sodium dodecyl sulfate; pH 7.2). 2 D epending on the method of analysis, do either of the following: I f you will use fluorescently-labeled antibodies (Procedure Va below), proceed directly to Step 3. If you will use peroxidase-conjugated antibodies (Procedure Vb below), then: Prior to hybridization, incubate fixed cells for 10 min at 0C with 1 mg/ ml of lysozyme in TE (100 mM TrisH C l, 50 mM ED TA; pH 8.0). Remove lysozyme by thoroughly rinsing the slide with sterile H 2O . Air dry the slide. Proceed to Step 3. 3 Add 8 l hybridization solution containing 50 ng of labeled oligonucleotide probe to the prepared slide (from Procedure II). 4 Incubate the slide for 2 h at 45C in an isotonically equilibrated humid chamber.
Va. Detection of DIG-labeled oligonucleotides with fluorescently labeled anti-DIG Fab fragments
N eofluar 100 x objective (oil immersion) on the following setup: a Zeiss Axioplan microscope fitted for epifluorescence microscopy with a 50 W mercury high pressure bulb and Zeiss filter sets 09 and 15 (Zeiss, O berkochen, FRG ). 6 For photomicrographs, use Kodak Ekta chrome P1600 color reversal film and an exposure time of 15 s for epifluorescence or 0.01 s for phase contrast micrographs.
Vb. Detection of DIG-labeled oligonucleotides with peroxidaseconjugated anti-DIG Fab fragments
1 U se the same conditions for hybridiza-
1 D ilute fluorescein- or rhodamine-labeled
anti-D IG Fab fragments 1:4 in blocking solution (150 mM sodium chloride; 100 mM Tris-H C l, pH 7.5; 0.5% bovine serum albumin; and 0.5% blocking reagent). 2 Add 10 l of the diluted antibody to the slide and incubate the slide for another hour at 27C in the humid chamber. 3 Remove the slide from the humid chamber and immerse in 40 ml of a washing solution (150 mM sodium chloride, 100 mM Tris-H Cl, 0.01% SDS; pH 7.4) at 29C for 10 min. 4 Prepare the slides for viewing by doing the following: Rinse the slides briefly with sterile water (which has been filtered through a 0.2 m filter). Air dry. Mount in C itifluor (C itifluor Ltd., London, U .K.).
tion and antibody binding as for fluorescent antibodies (Procedures IV and Va), except include a lysozyme pretreatment of the cell smears prior to hybridization. For details, see Procedure IV above. 2 Visualize the bound antibody as follows: Prepare peroxidase substrate-nickle chloride solution [1.3 mM diaminobenzidine, 0.02% (v/ v) H 2O 2, 0.03% (w/ v) nickel chloride, 5 mM Tris-H C l (pH 7.4)]. Incubate slide with peroxidase substrate-nickle chloride solution until a purplish-blue precipitate forms. 3 View slides under a light microscope and photograph with Kodak Ektachrome P1600 color reversal film.
Results
U sing the techniques in this article, we could detect P. cepacia cells in a mixed sample of three bacteria (Figure 1). Additional experiments with a D IG -labeled oligonucleotide not complementary to rRN A show no significant levels of nonspecifically bound D IG -labeled nucleic acid probes and anti-D IG antibodies.
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Figure 1: Specific identification . cepacia of whole fixed cells of P in a mixture of P . cepacia (chains of rods), E. coli (rods) and M. van-nielii (cocci) with a DIGlabeled oligonucleotide probe. Phase contrast (panel a) and epifluorescence (panel b) photos show detection with fluorescently labeled anti-DIG Fab fragments (Zarda et al., 1991). Phase contrast (panel c) and brightfield (panel d) photos show detection with peroxidase-conjugated anti-DIG Fab fragments
Reprinted from Zarda, B.; Amann, R.; W allner, G .; Schleifer, K. H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted en. Microbiol. 137, oligonucleotides. J. G 28232830 with permission of the society for general microbiology.
References
Amann, R. I.; Binder, B. J.; Olson, R. J.; Chisholm, S. W .; Devereux, R.; Stahl, D. A. (1990) Combination of 16 S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 19191925. Mhlegger, K.; Huber, E.; von der Eltz, H.; Rger, R.; Kessler, C. (1990) Nonradioactive labeling and detection of nucleic acids. Biol. Chem. Hoppe-Seyler 371, 953965. Reagent DIG-Oligonucleotide 3'-End Labeling Kit* DIG-Oligonucleotide 5'-End Labeling Set* Description 3'-end labeling of oligonucleotides from 14 to 100 nucleotides in length with DIG -11-ddUTP and terminal transferase With the DIGOligonucleotide 5'-End Labeling Set, oligonucleotides can be labeled with digoxigenin at the 5-end after synthesis that includes the addition of a phosphoramidite Fab Fragments from sheep Fab Fragments from sheep Fab Fragments from sheep Powder Zarda, B.; Amann, R.; W allner, G .; Schleifer, K. H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted oligonucleotides. J. G en. Microbiol. 137, 28232830.

Cat. No. 1 362 372 Pack size 1 Kit (25 labeling reactions) 1 Set (10 labeling reactions) 200 g 200 g 150 U 100 g 500 g 1 kg 250 g 500 g 1 kg 50 g 1g 10 g 100 g 500 g *Sold under the tradename of G enius in the US.
1 480 863
Anti-DigoxigeninFluorescein Anti-DigoxigeninRhodamine Anti-Digoxigenin-POD Tris
1 207 741 1 207 750 1 207 733 127 434 708 968 708 976 808 261 808 270 808 288 1 096 176 238 031 238 040 1 028 685 1 028 693
EDTA
Powder
Blocking Reagent BSA SDS
Powder Highest quality lyophilizate Purify 99%
CONTENTS
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Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections I SH to Tissues
Detection of HPV 11 DNA in paraffin-embedded laryngeal tissue with a DIG -labeled DNA probe
D r. J. R olighed and D r. H . L indeberg, EN T-departm ent and I nstitute for Pathology, A arhus U niv ersity H ospital, D enm ark .
The method is economical; the DIG
The technique of tissue in situ hybridization has become a powerful tool in pathology, although it is still mainly used for research purposes. The most important application at the moment is probably the demonstration of viral DN A (or RN A) in biopsies. This technique has made it possible to demonstrate infection with CMV (Grody et al., 1987; Unger et al., 1988), H BV (Blum et al., 1983; N egro et al., 1985), parvovirus (Proter et al., 1988), coxsackie B (Archard et al., 1987) EBV and H IV (Pezzella et al., 1989). In situ hybridization has been widely used to demonstrate H PV sequences in biopsies from the uterine cervix as well as from the head and neck (Lindeberg et al., 1990; Lindeberg et al., 1989; Shah et al., 1988). Although some of these viruses can be demonstrated by other methods, in situ hybridization is much faster than methods using infection of tissue cultures; for H PV tissue culture methods do not exist. Radioactively labeled probes were used originally, but in recent years nonradioactive probes have become increasingly popular for obvious reasons. In general, the use of radioactively labeled probes is considered superior to other methods. H owever, this is probably not correct (N uevo and Richart, 1989). For instance, H PV type 16 was demonstrated in SiH a cells by in situ hybridization with digoxigenin-labeled probes (H eiles et al., 1988). As SiH a-cells contain only 12 H PV copies per cell, one can hardly ask for a further improvement in sensitivity. There is an increasing demand from histopathologists for simple in situ hybridization methods, so that laboratory technicians can perform, for example, typing of H PV as a daily routine. To answer this demand, we present here a protocol for the detection of H PV DN A type 11 in laryngeal papillomas as well as in genital condylomatous lesions. The method is used on routine material which has been fixed in formalin and embedded in paraffin. The method described has the following advantages: The method is fast; results are obtained in about 56 h, and the hands-on time is about 2 h.
Labeling and Detection Kit* is sufficient for preparing 10 ml of DIG-labeled probe cocktail (1 l labeled DN A/ml). This is enough for hybridizing 1000 sections. Problems linked to endogenous biotin are avoided, since endogenous digoxigenin apparently does not exist. The main steps in performing in situ hybridization on formalin-fixed, paraffinembedded specimens are: 1 Exposure of target DN A. This is done with a carefully controlled proteolytic digestion step. 2 Denaturation of ds DN A, followed by hybridization. 3 Washing and detection of DN A hybrids. In our experience the most difficult step is optimization of the proteolytic digestion.
I. Probe preparation
1 Using standard methods, remove the viral
insert from its vector by restriction enzyme cleavage and separate it on a submerged agarose gel. 2 Recover the insert from the gel by electroelution or other methods. 3 Label the 8 kb viral insert nonradioactively with digoxigenin acccording to the procedures given in Chapter 4 of this manual. 4 To a tube, add the following ingredients of the probe cocktail: 10 l 50 x Denharts solution. 50 l dextran sulfate 50% (w/v). 10 l sonicated salmon sperm DN A (10 mg/l). 100 l 20 x SSC. 500 ng digoxigenin-labeled probe in 50 l TE. Enough distilled water to make a total volume of 250 l. 5 To complete the probe cocktail, add 250 l formamide to the tube. Tip: U se glov es w hen handling solutions containing form am ide. Form am ide should be handled in a fum e hood. 6 Mix the probe cocktail (by vortexing) and store it at 20C.
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Reagent DIG DNA Labeling and Detection Kit* , ** , *** Description Random primed labeling of DNA probes with DIG -11-dUTP , alkali-labile and color detection of DIG -labeled hybrids. Lyophilizate Cat. No. 1 093 657 Pack size 1 Kit (25 labeling reactions and detection of 50 blots of 100 cm2) 25 mg 100 mg 500 mg 1g 100 g 500 g 1 kg 250 g 500 g 1 kg 50 g 150 U (200 l) 8 ml
Proteinase K
161 519 745 723 1 000 144 1 092 766 127 434 708 968 708 976 808 261 808 270 808 288 1 096 176 1 093 274 1 681 451
Tris
Powder
EDTA
Powder
Blocking Reagent Anti-DIG-AP NBT/BCIP
Powder Fab Fragments from sheep Stock solution
***Sold under the tradename of G enius in the US. ***EP Patent 0 324 474 granted to Boehringer Mannheim G mbH. ***Licensed by Institute Pasteur.
References
Archard, L. C.; Bowles, N. E.; Olsen, E. G . J.; Richardson, P . J. (1987) Detection of persistent coxsachie B virus RNA in dilated cardiomyopathy and myocarditis. Eur. Heart J. (Suppl.) 8, 437440. Blum, H. E.; Stowringer, L.; Figue, A. et al. (1983) Detection of Hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization. Proc. Natl. Acad. Sci. (USA) 80, 66856688. G rody, W .W .; Cheng, L.; Lewin, K. J. (1987) In situ viral DNA hybridization in diagnostic surgical pathology. Hum. Pathol. 18, 535543. Heiles, H. B. J.; G enersch, E.; Kessler, C.; Neumann, R.; Eggers, H. J. (1988) In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells. BioTechniques 6 (10), 978981. Lindeberg, H.; Johansen, L. (1990) The presence of human papillomavirus (HPV) in solitary adult laryngeal papillomas demonstrated by in situ DNA hybridization with sulphonated probes. Clin. Otolaryngol. 15, 367371. Lindeberg, J.; Syrjnen, S.; Karja, J.; Syrjnen, K. (1989) Human papillomavirus type 11 DNA in squamous cell carcinomas and preexisting multiple laryngeal papillomas. Acta Oto-Laryngol. 107, 141149. Negro, F .; Berninger, M.; Chaiberge, E. et al. (1985) Detection of HBV-DNA by in situ hybridization using a biotin-labeled probe. J. Med. Virol. 15, 373382. Nuevo, G . J.; Richart, R. M. (1989) A comparison of Biotin and 35S-based in situ hybridization methodologies for detection of human papillomavirus DNA. Lab. Invest. 61, 471475. Pezzella, M.; Pezzella, F .; Rapicetta, M. et al. (1989) HBV and HIV expression in lymph nodes of HIV positive LAS patients: histology and in situ hybridization. Mol. Cell Probes 3, 125132. Proter, H. J.; Quantrill, A. M.; Fleming, K. A. (1988) B19 parvovirus infection of myocardial cells. Lancet 1, 535536. Shah, K. V .; G upta, J. W .; Stoler, M. H. (1988) The in situ hybridization test in the diagnosis of human papillomaviruses. In: De Palo, G .; Rilke, F .; zur Hausen, H. (Eds.) Serono Symposia 46: Herpes and Papillomaviruses. New Y ork: Raven Press, 151159. Unger, E. R.; Budgeon, L. R.; Myorson, B.; Brigati, D. J. (1988) Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method. Am J. Surg. Pathol. 18, 18.
CONTENTS
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125
II. Pretreatment of slides

1 Wash slides overnight in a detergent, fol-
IV . Hybridization
1 After the treatment with Proteinase K,
lowed by an intensive wash in tap water, and finally, a wash in demineralized water. 2 After drying the slides, wash them for 3 min in acetone. 3 Transfer the washed slides to a 2% solution of 3-aminopropyltrithoxysilane in acetone and incubate for 5 min. 4 Wash the slides briefly in distilled water and allow to dry. N ote: T he coated slides can be stored dry under dust-free conditions for sev eral m onths.
remove the coverslips and fix the sections in 0.4% formaldehyde for 5 min at 4C. 2 Immerse the sections in distilled water for 5 min. 3 Allow the sections to drip and air dry for 5 min. 4 Distribute about 510 l of the probe cocktail over each section. N ote: L arge biopsies m ay require larger v olum es of probe cock tail.
5 Prepare a negative control by covering
III. Preparation of tissue sections

1 Fix biopsies from condylomatous lesions
and from laryngeal papillomas in 4% phosphate-buffered formaldehyde and embed them in paraffin. 2 Cut routine sections, float them on demineralized water and place them on the pretreated slides. 3 Treat the sections on the slides as follows: Bake the sections for 30 min at 60C. Dewax in xylene. Rehydrate them by dipping them in a graded ethanol series (2 x in 99% ethanol, 2 x in 96% ethanol, 1x in 70% ethanol, 1x in H 2O ). 4 Immediately before use, prepare a working Proteinase K solution as follows: Thaw an aliquoted frozen stock solution of Proteinase K, 10 mg/ml, in H 2O . Dilute the stock Proteinase K to the working concentration of 100 g/ml in TES [50 mM Tris-H Cl (pH 7.4), 10 mM EDTA, 10 mM N aCl]. 5 Treat each section with 2030 l of the working solution of Proteinase K for 15 min at 37C. During the proteolytic digestion cover the sections with coverslips and place them in a humid chamber. Tip: We use only siliconiz ed cov erslips. N ote: T he proteolytic treatm ent is crucial and m ay need to be v aried w ith different tissues.W hen too m uch Proteinase K is used, the tissue totally disintegrates, and if too little is used positiv e signals m ay be totally absent.
one section from each block with a blind probe cocktail, i.e. a probe cocktail containing all the ingredients in Procedure I, except the labeled H PV DN A. 6 Place coverslips over each section and denature the DN A by placing the slides on a heating plate at 95C for 6 min. Tip: We use only siliconiz ed cov erslips.
7 Cool the slides for 1 min on ice. 8 Place the slides in a humid chamber and
incubate for 3 h at 42C in an oven. Tip: T he hybridiz ation tim e m ay be prolonged ov ernight w hen conv enient.
V .W ashes
Remove the coverslips and wash the sections as follows:
2 x 5 min in 2 x SSC at 20C. 1 x 10 min in 0.1 x SSC at 42C.
CONTENTS
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VI. Detection of hybrids

1 D ip each section into buffer 1 [100 mM
2 Cover each section with 2040 l buffer 2
Tris-H C l, 150 mM N aC l;pH 7.5 (20C )].
10 After the incubation, do the following: Remove the coverslips. Wash the sections gently. Stain a few seconds with neutral red. Mount.
[0.5% (w/v) blocking reagent in buffer 1], then add a coverslip. 3 Incubate sections at 20C for 15 min. 4 Remove the coverslips and dip the sections in buffer 1. 5 Dilute the antibody conjugate 1:500 in buffer 2. 6 Distribute 1020 l of the diluted con jugate over each section, including the negative controls. 7 Place a coverslip over each section and place all sections in a humid chamber at room temperature for 1 h. 8 Remove the coverslips, wash the sections 2 x10 min in buffer 1, then equilibrate for 5 min in buffer 3. 9 Distribute approx. 20 l color-solution (N BT/BCIP) onto each section, cover with a coverslip and leave in the dark overnight. N ote: You m ay shorten the colorreaction to 3060 m in if you w ish. I n fact, a positiv e reaction m ay be seen after a few m inutes in the dark w hen H PV is present in a high copy-num ber. Hint: U se glov es w hen handling the color solution.
C aution: A v oid dehydrating procedures since ethanol m ay rem ov e or w eak en the signal.
Results
The described method is applied to biopsies of multiple and solitary laryngeal papillomas and on a flat condylomatous lesion of the uterine cervix (Figure 1). The positive results are obvious, and little background is observed in these examples. The procedure is extremely straightforward and can be performed in 56 h, including the 3 h hybridization. Consequently, the whole procedure can be performed in 1 day and the results recorded the next morning. If needed, the hybridization can be shortened; in our initial experiments, we were able to detect H PV type 11 after only 1 h of hybridization.
Acknowledgment
Plasmids with H PV type 11 were kindly supplied by Prof. zur H ausen and co-workers, DKFZ, H eidelberg, Germany.
Figure 1: Detection of HPV 11 DNA in biopsies of multiple and solitary laryngeal papillomas and on a flat condylomatous lesion of the uterine cervix. Positive results are shown in Panels a and c. The negative controls (Panels b and d) show no signal.
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Detection of mRNA in tissue sections using DIG -labeled RNA and oligonucleotide probes
P. Kom m inoth, D iv ision of C ell and M olecular Pathology, D epartm ent of Pathology, U niv ersity of Z rich, Sw itz erland.
The protocols given below are modifications of recently published methods for nonisotopic in situ hybridization with digoxigenin-labeled probes (Komminoth, 1992; Komminoth et al., 1992). In those reports we demonstrated:
Digoxigenin-labeled probes are as sensi Protocols with digoxigenin-labeled probes
tive as 35S-labeled probes.
The protocols below are written in general terms. Additional information concerning the in situ hybridization methods and important technical details are provided in Komminoth (1992), Komminoth et al. (1992), Komminoth et al. (1995), Sambrook et al. (1989), and the Boehringer Mannheim DIG/Genius System Users Guide for Membrane H ybridization.
may also be applied to diagnostic in situ hybridization procedures. The following protocols have been successfully used in our laboratories to detect mRN A in frozen and paraffin-embedded tissue sections with either RN A or oligonucleotide probes. RN A probes are best for high sensitivity detection procedures because:
RN A probes are easily generated and
I. Preparation of slides
N ote: G elatin-coated slides are excellent for large tissue sections obtained from froz en or paraffin-em bedded specim ens. A lternativ ely, silane-coated slides can be used, but are better suited for sm all tissue sam ples or cell preparations.
IA. Gelatin-coated slides
1 Clean slides by soaking for 10 min in
2 Wash slides in running hot water. 3 Rinse slides in distilled water. 4 Prepare gelatin solution as follows: Dissolve 10 g gelatin (Merck) in 1 liter
labeled by in vitro transcription procedures. H ybrids between mRN A and RN A probes are highly stable. Protocols using stringent washing conditions and RN ase digestion steps yield highly specific signals with low background. Synthetic oligonucleotide probes are an attractive alternative to RN A probes for the detection of abundant mRN A sequences in tissue sections, because:
Any known nucleic acid sequence can
Chromerge solution (Merck).
rapidly be made by automated chemical synthesis. Such probes are more stable than RN A probes. O ligonucleotide probes are labeled during synthesis or by addition of reporter molecules at the 5' or 3' end after synthesis. The most efficient labeling method is addition of a tail of labeled nucleotides to the 3' end. O ligonucleotide probes are generally less sensitive than RN A probes since fewer labeled nucleotides can be incorporated per molecule of probe.
of distilled water which has been heated to 4050C. Add 4 ml 25% chromium potassium sulfate (CPS) solution (Merck) to the gelatin to make a final concentration of 0.1% CPS. 5 Dip slides in gelatin solution for 10 min. 6 Let the slides air dry. 7 Soak slides for 10 min in PBS (pH 7.4) containing 1% paraformaldehyde. 8 Let the slides air dry. 9 Bake slides at 60C overnight.
IB. Silane-coated slides
1 Soak clean glass slides for 60 min in silane
solution [5 ml of 3-aminopropyltriethoxysilane (Sigma) in 250 ml of acetone]. 2 Wash the slides 2 x 10 min in distilled water. 3 Dry slides overnight at 60C. N ote: Both gelatin- and silane-coated slides can be stored for sev eral m onths under dry and dust free conditions.
126
CONTENTS
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II. Tissue preparation

General guidelines: Fix or freeze tissue as soon as possible after surgical excision to prevent degradation of mRN A. If possible, use cryostat sections of paraformaldehyde-fixed tissues which have been immersed in sucrose (as in Procedure IIA). These provide excellent conditions for mRN A localization and preservation of sample morphology. H owever, in surgical pathology, most tissues are routinely fixed in formalin. For mRN A localization, do not fix in formalin for more than 24 h. Prolonged storage of samples in formalin will cause covalent linkages between mRN A and proteins, making target sequences less accessible. C aution: Prepare all solutions for procedures I I A and I I B w ith distilled, deioniz ed w ater (ddH 2O ) that has been treated w ith 0.1% diethylpyrocarbonate (D EPC ) (Sam brook et al., 1989).
IIA. Frozen sections
1 Cut tissues into 2 mm thick slices. 2 Incubate cut tissues at 4C for 24 h with
IIB. Paraffin sections

1 Prepare formaldehyde-fixed and paraffin-
embedded material according to standard procedures. 2 Cut 7 m sections from the paraffin embedded material and place the sections onto coated glass slides (from Procedure I). 3 Dry slides in an oven at 40C overnight. 4 Dewax sections 2 x 10 min with fresh xylene. 5 Rehydrate sections in the following solutions: 1 x 5 min in 100% ethanol. 1 x 5 min in 95% ethanol. 1 x 5 min in 70% ethanol. 2 x in DEPC-treated ddH 2O .
IIIA. ISH protocol for detection of mRNA with DIG -labeled RNA probes
C aution: Prepare all solutions for procedures below (probe labeling through posthybridiz ation) w ith distilled, deioniz ed w ater (ddH 2O ) that has been treated w ith 0.1% diethylpyrocarbonate (D EPC ) (Sam brook et al., 1989). To av oid R N ase contam ination, w ear glov es throughout the procedures and use different glassw are for pre- and posthybridiz ation steps. N ote: Perform all procedures below at room tem perature unless a different tem perature is stated. Probe labeling N ote: To ensure tissue penetration, w e prefer to w ork w ith R N A probes that are ! 500 bases long.
1 Clone a cDN A insert into an RN A
freshly made, filtered fixative (DEPCtreated PBS containing 4% paraformaldehyde; pH 7.5). 3 Decant fixative and soak tissues at 4C overnight in sucrose solution [DEPCtreated PBS containing 30% sucrose (RN ase-free)]. N ote: D uring this step, the tissues should sink to the bottom of the container.
4 Store tissue samples in a freezing com
pound at 80C, or, for long time storage, at 140C (N aber et al., 1992). 5 For sectioning, warm the samples to 20C and cut 10 m sections in a cryostat. 6 Place the sections on pretreated glass slides (from Procedure I). 7 Dry slides in an oven at 40C overnight. 8 Do either of the following: Use the prepared slides immediately. or Store the slides in a box at 80C. Before processing, warm the stored slides to room temperature and dry them in an oven at 40C for a minimum of 2 h. N ote: Slides w ith cryostat sectionsm ay be stored at 80 C for sev eral w eek s.
expression vector (plasmid) according to standard procedures (Sambrook et al., 1989). 2 Linearize the RN A expression vector with appropriate restriction enzymes to allow in vitro run-off synthesis of both sense- and antisense-oriented RN A probes (Valentino et al., 1987). N ote: A s an alternativ e to lineariz ed plasm ids, use PC R -generated tem plates containing R N A polym erase prom oter sequences for in v itro transcription (Young et al., 1991).
CONTENTS
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127
3 Purify linearized plasmid by phenol-
chloroform extraction and ethanol precipitation. C aution: Som e plasm id purification k its contain R N ase digestion steps for rem ov ing bacterial R N A . Traces of R N ase m ay destroy transcribed probes. To ensure rem ov al of residual R N ase, perform m ultiple phenol-chloroform extractions of the lineariz ed plasm id.
4 To avoid RN A polymerase inhibition,
resuspend the plasmid in EDTA-free buffer or DEPC-treated ddH 2O . 5 Generate digoxigenin-labeled RN A probes in both the sense and antisense direction by in vitro transcription with the DIG RN A Labeling Kit* or according to procedures in Chapter 4 of this manual. 6 Purify labeled probes according to procedures in the DIG/Genius System Users Guide for Membrane H ybridization or in Chapter 4 of this manual. C aution: Probes longer than 500 bases m ay not penetrate tissue. I f probes are longer than 500 bases, shorten them by alk aline hydrolysis according to procedures in the D I G / G enius System U sers G uide for M em brane H ybridiz ation.
7 Estimate the yield of labeled probes by
C aution: Perm eabiliz ation is the m ost critical step of the entire in situ hybridiz ation procedure. I n paraffin-em bedded archiv al m aterials, optim al tissue perm eabiliz ation differs for each case, depending upon duration and type of fixation. We recom m end titration of the Proteinase K concentration. A lternativ e perm eabiliz ation protocols for im prov ed efficiency of digestion include incubation of slides for 2030 m in at 37C w ith 0.1% pepsin in 0.2 M H C l.
5 Post-fix sections for 5 min at 4C with
DEPC-treated PBS containing 4% paraformaldehyde. 6 Wash sections 2 x 5 min with DEPC treated PBS. 7 To acetylate sections, place slide containers on a rocking platform and incubate slides 2 x 5 min with 0.1 M triethanolamine (TEA) buffer, pH 8.0, containing 0.25% (v/v) acetic anhydride (Sigma). C aution: A cetic anhydride is highly unstable. A dd acetic anhydride to each change of T EA -acetic anhydride solution im m ediately before incubation.
8 Incubate sections at 37C for at least
direct blotting procedures as described in Chapter 4 of this manual. 8 Aliquot labeled probes and store them at 80C. N ote: Probes are stable for up to one year. Prehybridization
1 Incubate sections as follows: 2 x 5 min with DEPC-treated PBS (pH
10 min with prehybridization buffer [4 x SSC containing 50% (v/v) deionized formamide]. N ote: D eioniz e form am ide w ith D ow ex M R 3 (Sigm a) according to protocols described in Sam brook et al. (1989). N ote: 1 x SSC = 150 m M N aC l, 15 m M sodium citrate, pH 7.2. In situ hybridization
1 Prepare hybridization buffer containing: 40% deionized formamide. 10% dextran sulfate. 1 x Denhardts solution [0.02% Ficoll,
7.4) (Sambrook et al., 1989). N ote: PBS contains 140 m M N aC l, 2.7 m M KC l, 10 m M N a2H PO 4, 1.8 m M KH 2PO 4. 2 x 5 min with DEPC-treated PBS containing 100 mM glycine. 2 Treat sections for 15 min with DEPC treated PBS containing 0.3% Triton X-100. 3 Wash 2 x 5 min with DEPC-treated PBS. 4 Permeabilize sections for 30 min at 37C with TE buffer (100 mM Tris-H Cl, 50 mM EDTA, pH 8.0) containing either 1 g/ml RN ase-free Proteinase K (for frozen sections) or 520 g/ml RN asefree Proteinase K (for paraffin sections).
0.02% polyvinylpyrrolidone, 10 mg/ml RN ase-free bovine serum albumin]. 4 x SSC. 10 mM DTT. 1 mg/ml yeast t-RN A. 1 mg/ml denatured and sheared salmon sperm DN A. C aution: D enature and add salm on sperm D N A to buffer shortly before hybridiz ation. N ote: H ybridiz ation buffer (m inus salm on sperm D N A ) can be stored at 20 C for sev eral m onths.
128
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2 Drain prehybridization buffer from the
1 Using a shaking platform, wash sections
slides and overlay each section with 30 l of hybridization buffer containing 510 ng of digoxigenin-labeled RN A probe. 3 Cover samples with a 24 x 30 mm hydrophobic plastic coverslip (e.g. cut from Gel Bond Film, FMC BioProducts, Rockland, ME, USA). 4 Incubate sections at 42C overnight in a humid chamber. Posthybridization C aution: U se a separate set of glassw are for posthybridiz ation and prehybridiz ation procedures to av oid R N ase contam ination in prehybridiz ation steps.
1 Remove coverslips from sections by
2 x 10 min with buffer 1 [100 mM TrisH Cl (pH 7.5), 150 mM N aCl]. 2 Cover sections for 30 min with blocking solution [buffer 1 containing 0.1% Triton X-100 and 2% normal sheep serum (Sigma)]. 3 Decant blocking solution and incubate sections for 2 h in a humid chamber with buffer 1 containing 0.1% Triton X-100, 1% normal sheep serum, and a suitable dilution of sheep anti-DIG-alkaline phosphatase [Fab fragments]. N ote: For optim al detection, incubate sev eral sections (from the sam e sam ple) w ith different dilutions of the antibody (1:100; 1:500, and 1:1000).
4 Using a shaking platform, wash sections 5 Incubate sections for 10 min with buffer 2
immersing slides for 510 min in 2 x SSC. C aution: D o not place sam ples w hich are hybridiz ed w ith different probes in the sam e container.
2 In a shaking water bath at 37C, wash
2 x10 min with buffer 1.
sections as follows: 2 x15 min with 2 x SSC. 2 x15 min with 1x SSC. 3 To digest any single-stranded (unbound) RN A probe, incubate sections for 30 min at 37C in N TE buffer [500 mM N aCl, 10 mM Tris, 1 mM EDTA, pH 8.0] containing 20 g/ml RN ase A. C aution: Be particularly careful w ith R N ase. T his enz ym e is extrem ely stable and difficult to inactiv ate. A v oid contam ination of any equipm ent or glassw are w hich m ight be used for probe preparation or prehybridiz ation procedures. U se separate glassw are for R N ase-contam inated solutions.
sections 2 x 30 min with 0.1 x SSC. N ote: I f this procedure giv es high back ground or nonspecific signals, try posthybridiz ation w ashings at 52 C w ith 2 x SSC containing 50% form am ide. Immunological detection N ote: T his detection procedure uses com ponents of the D I G N ucleic A cid D etection Kit*. A lternativ e detection procedures include the im m unogold m ethod w ith silv er enhancem ent for enz ym e-independent probe detection (Kom m inoth et al. 1992; Kom m inoth et al., 1995) and other detection procedures described in C hapter 5 of this m anual.
[100 mM Tris-H Cl (pH 9.5), 100 mM N aCl, 50 mM MgCl2]. 6 Prepare a color solution containing: 10 ml of buffer 2. 45 l nitroblue tetrazolium (N BT) solution (75 mg N BT/ml in 70% dimethylformamide). 35 l 5-bromo-4-chloro-3-indolylphosphate (BCIP or X-phosphate) solution (50 mg X-phosphate/ml in 100% dimethylformamide). 1 mM (2.4 mg/10 ml) levamisole (Sigma). N ote: For conv enience, prepare a 1 M stock solution of lev am isole in ddH 2O (stable for sev eral w eek s, w hen stored at 4C ) and add 10 l of the stock to 10 m l of the color solution. I f endogenous phosphatase activ ity is high, increase the concentration of lev am isole to 5 m M (50 l 1 M stock / 10 m l solution) in the color solution. N ote: N BT / BC I P produces a blue precipitating product. For other colors, use other alk aline phosphatase substrates. For exam ple, use either Fast R ed or I N T / BC I P for red/ brow n precipitates.
7 Cover each section with approximately
200 l color solution and incubate slides in a humid chamber for 224 h in the dark. 8 When color development is optimal, stop the color reaction by incubating the slides in buffer 3 [10 mM Tris-H Cl (pH 8.1), 1 mM EDTA]. 9 Dip slides briefly in distilled water.
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129
10 Counterstain sections for 12 min with
3 Add a 24 x 30 mm coverslip to each
0.02% fast green FCF or 0.1% nuclear Fast Red (Aldrich Chemical Corp., Milwaukee, WI, USA) in distilled H 2O . 11 Wash sections 2 x 10 min in tap water. 12 Mount sections using an aqueous mounting solution (e.g. Aqua-Mount from Lerner Laboratories, Pittsburgh, PA, USA). C aution: D o not use xylene-based m ounting solutions. T hese lead to crystal form ation of color precipitates.
section and incubate slides in a humid chamber at 37C for 2 h. 4 Remove coverslips by immersing slides for 5 min in 2xSSC. In situ hybridization N ote: To increase the sensitiv ity of in situ hybridiz ation procedures, use m ixtures of oligonucleotide probes that are com plem entary to different regions of the target RN A.
1 Prepare hybridization buffer containing: 2 x SSC. 1 x Denhardts solution [0.02% Ficoll,
IIIB. ISH protocol for detecting mRNA with DIG -labeledoligonucleotideprobes

Probe labeling
1 Label 100 pmol of oligonucleotide probe
(2030 mer) by tailing with the DIG O ligonucleotide Tailing Kit* according to the protocol described in Chapter 4 of this manual. 2 Purify labeled probes according to pro cedures in the DIG/Genius System Users Guide for Membrane H ybridization or in Chapter 4 of this manual. 3 Estimate the yield of labeled probes by direct blotting procedures as described in Chapter 4 of this manual. 4 Aliquot the labeled probes and store them at 20C. N ote: Probes are stable for up to one year. N ote: To increase the sensitiv ity of in situ hybridiz ation procedures, prepare sev eral labeled probes that are com plem entary to different regions of the target R N A , then use m ixtures of the probes for the hybridiz ation step below. Prehybridization
1 Follow Steps 17 from the prehybridi-
0.02% polyvinylpyrrolidone, 10 mg/ml RN ase-free bovine serum albumin]. 10% dextran sulfate. 50 mM phosphate buffer (pH 7.0). 50 mM DTT. 250 g/ml yeast t-RN A. 5 g/ml polydeoxyadenylic acid. 100 g/ml polyadenylic acid. 0.05 pM/ml Randomer O ligonucleotide H ybridization Probe (DuPont). 500 g/ml denatured and sheared salmon sperm DN A. C aution: D enature and add salm on sperm D N A to buffer shortly before hybridiz ation.
Enough deionized formamide (% dF,
as calculated by the formula below) to produce stringent hybridization conditions at 37C [that is, to make 37C = T m (oligonucleotide probe) 10C] (Long et al., 1992). C aution: % dF should not exceed 47% of the total v olum e of the hybridiz ation buffer. I f dF is less than 47% , add ddH 2O so that ddH 2O plus dF equals 47% of the v olum e. N ote: U se the follow ing form ula to calculate the form am ide concentration (% dF) in the hybridiz ation buffer for each oligonucleotide probe:
zation section of Procedure IIIA (for RN A probes). 2 O verlay each section with 40 l prehybridization buffer (identical to the hybridization buffer to be used for in situ hybridization below, but containing no labeled probe).
(7.9) + 81.5 + 0.41 (% GC) 675/L % mismatch (47) _ % dF = ______________________________________________ 0.65 where, in this case: 7.9 = 16.6 log10 [N a+] (for 2 x SSC) % GC = % [G + C] base content of the oligonucleotide L = oligonucleotide length (in bases) % mismatch = % (bases in oligonucleotide not complementary to target) 47 = hybridization temperature + 10C (for 37C)
130
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N ote: H ybridiz ation buffer (m inus dF, ddH 2O , and salm on sperm D N A ) can be stored at 20 C for sev eral m onths.
2 Drain 2 x SSC (Step 4, Prehybridization)
N egative controls
1 N egative sample: Tissue or cell line
2 Technical controls: Target: Digestion of mRN A with
known to lack the sequence of interest.
from the slides and overlay each section with 30 l of hybridization buffer containing 1030 ng of digoxigeninlabeled oligonucleotide probe. 3 Cover sections with a 24 x 30 mm hydrophobic plastic coverslip (e.g. cut from Gel Bond Film, FMC BioProducts, Rockland, ME, USA). 4 Incubate samples at 37C overnight in a humid chamber. Posthybridization
1 Remove coverslips from sections by
immersing slides for 510 min in 2 x SSC. sections as follows: 2 x 15 min with 2 x SSC. 2 x 15 min with 1x SSC. 2 x 15 min with 0.25 x SSC.
RN ase prior to in situ hybridization (should give negative results). H ybridization: H ybridization with sense probe H ybridization with irrelevant probe (e.g. probe for viral sequences) H ybridization in the presence of excess unlabeled antisense probe (all should give negative results). Detection: H ybridization without probe O mission of anti-DIG antibody (both should give negative results).
Results
Immunological detection Use the same immunological detection protocol as in Procedure IIIA (for RN A probes) above.
Controls for in situ hybridizations
Adequate controls must always be included to ensure the specificity of detection signals. Controls should include positive and negative samples as well as technical controls to detect false positive and negative results (H errington and McGee, 1992). Positive controls
1 Positive sample: Tissue or cell line known
2 Technical control to test quality of tissue
5
A B
to contain mRN A of interest.
mRN A and the procedure reagents: Labeled poly(dT) probe (for oligonucleotide in situ hybridization only); labeled RN A or oligonucleotide probes complementary to abundant housekeeping genes (such as -tubulin) to test quality of tissue mRN A and the procedure reagents (should give positive results).
Figure 1: Comparison of 35S- and digoxigenin-labeled cRNA probes for the detection of seminal vesicle secretion protein II (SVS II) mRNA in cryostat sections of the dorsolateral rat prostate. Note the equal intensity of signals in acini of the lateral lobe obtained with isotopic (Panel A) and non-isotopic (Panel B) in situ hybridization procedures. Also note the absence of signal in the coagulating glands (arrows in Panel B) which serves as an internal negative control.
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131
Figure 2: SVS II mRNA detection in contiguous glands of the rat prostate with a digoxigeninlabeled antisense RNA probe. Panel A: Strong signals are present in epithelia of the ampullary gland and no signals are encountered in adjacent duct epithelia of the coagulating gland (arrow). Panel B: A higher magnification shows that the hybridization signal is restricted to the cytoplasmic portion of the glandular epithelial cells (cryostat sections).
5
B
Figure 3: SVS II mRNA detection in contiguous

glands of the rat prostate with a digoxigeninlabeled oligonucleotide antisense probe. Hybridization signals appear to be slightly less strong than with the SVS II RNA probe (Figure 2).
132
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Figure 4: Detection of parathyroid hormone (PTH) mRNA in formaldehyde-fixed, paraffinembedded tissue of a parathyroid gland with a digoxigenin-labeled antisense RNA probe. Panel A: Note the weaker signal in cells of an adenomatous lesion (upper right part) compared with the normal parathyroid tissue. Panel B: Higher magnification shows the excellent resolution of hybridization signals, which are confined to the cytoplasmic portion of cells.
A Figure 5: Use of a digoxigenin-labeled antisense RNA probe and Fast Red chromogen to detect PTH mRNA in formaldehyde-fixed, paraffinembedded tissue of a parathyroid gland with chief cell hyperplasia. Panel A: PTH mRNA is marked by red precipitates. Panel B: Only weak signals are present when a sense PTH probe is used as a control.
5
A Figure 6: Detection of synaptophysin mRNA in formaldehyde-fixed, paraffin-embedded tissue of a neuroendocrine carcinoma of the gut with digoxigenin-labeled oligonucleotide probes and immunogold-silver enhancement method for visualization. Note the strong hybridization signals (consisting of silver precipitates) over tumor cells in Panel A (where an antisense probe was used) and the much weaker signal in Panel B (where the appropriate sense probe was used as a control). B
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133

Reagent DIG RNA Labeling Kit* , ** , *** DIG Oligonucleotide Tailing Kit* , *** , **** DIG Nucleic Acid Detection Kit* Description For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase. For tailing oligonucleotides with digoxigenin-dUTP . For detection of digoxigenin-labeled nucleic acids by an enzyme-linked immunoassay with a highly specific antiDIG -AP antibody conjugate and the color substrate NBT and BCIP . Vicous, liquid Powder Cat. No. 1175 025 Pack size 1 Kit (2x10 labeling reactions) 1 Kit (25 tailing reactions) 1 Kit
1 417 231 1175 041
Triton X-100 EDTA
789 704 808 261 808 270 808 288 1 413 783 1 373 196 1 373 200 108 057 1 693 387 238 031 238 040 197 777 708 984 1 583 786 708 992 709 000 127 434 708 968 708 976 109 142 109 169 109 495 109 509 108 626 223 581
100 ml 250 g 500 g 1 kg 1.25 ml 5 ml 25 ml 1g 5g 1g 10 g 2g 10 g 25 g 50 g 100 g 100 g 500 g 1 kg 25 mg 100 mg 100 mg 500 mg 100 mg 5 A260 units
Proteinase K
Solution
Pepsin BSA DTT
Lyophilizate Highest quality, lyophilizate Purity: > 97%
Tris
Powder
RNase A tRNA Poly(A) Poly(dA)
From bovine pancreas, dry powder From bakers yeast, lyophilizate Polyadenylic acid Poly-deoxy-adenylic acid
****Sold under the tradename of G enius in the US. ****EP Patent 0 324 474 granted to Boehringer Mannheim G mbH. ****Licensed by Institute Pasteur. ****EP Patents 0 124 657/0 324 474 granted to Boehringer Mannheim G mbH.
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Acknowledgments
I am grateful to Ph. U. H eitz and J. Roth for general support; to A. A. Long, H . J. Wolfe, S. P. N aber, and W. Membrino for technical advice; to M. Machado, X. Matias-Guiu, F. B. Merck, I. Leav, and P. Saremaslani for technical support; and to N . Wey and H . N ef for photographic reproductions. The projects during which the protocols have been established were supported in part by the Julius Mller-Grocholski Cancer Research Foundation, Zrich Switzerland.
References
Herrington, C. S.; McG ee, J. O. (1992) Principles and basic methodology of DNA/RNA detection by in situ hybridization. In: Herrington, C. S.; McG ee, J. O. (Eds.) Diagnostic molecular pathology: ork: IRL Press, Vol. 1, A practical approach. New Y pp. 69102. Komminoth, P . (1992) Digoxigenin as an alternative probe labeling for in situ hybridization. Diagn. Mol. Pathol. 1, 142150. Komminoth, P .; Merk, F . B.; Leav, I.; W olfe, H. J.; Roth, J. (1992) Comparison of 35S- and digoxigeninlabeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate. Histochemistry 98, 217228. Komminoth, P .; Roth, J.; Saremaslani, P .; Schrdel, S., Heitz, P . U. (1995) Overlapping expression of immunohistochemical markers and synaptophysin mRNA in pheochromocytomas and adrenocortical carcinomas. Implications for the differential diagnosis of adrenal gland tumors. Lab. Invest. 72, 424431. Long, A. A.; Mueller, J.; Andre-Schwartz, J.; Barrett, K.; Schwartz, R., W olfe, H. J. (1992) High-specificity in situ hybridization: Methods and application. Diagn. Mol. Pathol. 1, 4557. Naber, S.; Smith, L.; W olfe, H. J. (1992) Role of the frozen tissue bank in molecular pathology. Diagn. Mol. Pathol. 1, 7379. Sambrook, J.; Fritsch, E.; Maniatis, T . (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press. Valentino, K. L.; Eberwine, J. H.; Barchas, J. D. (1987) In situ Hybridization: Applications To Neurobiology. Oxford, England: Oxford University Press. Y oung, I. D.; Ailles, L.; Deugau, K.; Kisilevsky, R. (1991) Transcription of cRNA for in situ hybridization from Polymerase Chain Reaction-amplified DNA. Lab. Invest. 64, 709712.
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135
Detection of mRNA on paraffin embedded material of the central nervous system with DIG -labeled RNA probes
H . Breitschopf and G . Suchanek , R esearch U nit for Experim ental N europathology, A ustrian A cadem y of Sciences, V ienna, A ustria.
4 Coat slides with a 2% solution of
The following protocol was developed to show mRN A in paraffin embedded material of the central nervous system. It also allows double staining of protein and mRN A. This method is sensitive enough to show mRN A expressed at very low levels (Breitschopf et al., 1992).
3-aminopropyltriethoxy-silane (Sigma) in acetone, then air dry them. C aution: Perform Step 4 under R N asefree conditions.
5 Cut 4 m thick sections from the fixed
I. Probe preparation
1 Prepare plasmids by standard methods
samples with disposable knifes and attach them to the prepared slides. C aution: Perform Step 5 under R N asefree conditions.
6 Dry the sections overnight at 55C. 7 Store the sample slides at room temper-
(Sambrook et al., 1989). 2 Prepare RN A probes from the plasmids and label them with digoxigenin (DIG) either according to the methods in Chapter 4 of this manual or according to the instructions in the DIG RN A Labeling Kit*. 3 Remove unincorporated nucleotides from the labeled probes by passing the labeling mixture over a Q uick Spin column for radiolabeled RN A preparation (Sephadex G-50). C aution: O m ission of this colum n step m ay lead to back ground problem s.
4 Determine the amount of DIG-labeling
ature until they are used.
III. Pretreatment of sections

C aution: Perform pretreatm ent and hybridiz ation steps under R N ase-free conditions. Prepare all solutions and buffers for these steps w ith D EPC -treated w ater (Sam brook et al., 1989). Bak e all glassw are for 4 h at 180 C . A ssum e that disposable plasticw are is R N ase-free.
1 Dewax slides extensively by treating with
with a dot blot according to the method in Chapter 4 of this manual.
II. Tissue preparation

1 U se samples fixed by standardized
methods, such as: Perfusion-fixed samples. C aution: Before using perfusion-fixed samples, treat them further by immersion-fixing for 34 h at room temperature in 100 mM phosphate buffer containing 4% paraformaldehyde. N ote: Sam ples fixed w ith 4% paraform aldehyde giv e the best results in this procedure. Routinely fixed autopsy samples. Samples fixed with 2.5% glutaraldehyde. 2 After fixation, wash the samples in phosphate buffered saline (PBS). 3 Embed the fixed samples in paraffin.
xylene (preferably overnight) and a graded series of ethanol solutions. 2 To preserve the mRN A during the following procedure, fix the sections once again with 4% paraformaldehyde (in 100 mM phosphate buffer) for 20 min. 3 Rinse the sections 35 times with TBS buffer [50 mM Tris-H Cl (pH 7.5); 150 mM N aCl]. 4 Treat the sections for 10 min with 200 mM H Cl to denature proteins. 5 Rinse the sections 35 times with TBS. 6 To reduce nonspecific background (H a yashi et al., 1978), incubate the sections for 10 min on a magnetic stirrer with a freshly mixed solution of 0.5% acetic anhydride in 100 mM Tris (pH 8.0). 7 Rinse the sections 35 times with TBS. 8 Treat the sections for 20 min at 37C with Proteinase K (10500 g per ml in TBS which contains 2 mM CaCl2). The concentration of Proteinase K depends upon the degree of fixation. Generally, start with 20 g/ml Proteinase K
136
CONTENTS
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for paraformaldehyde-fixed tissue. The concentration needed for glutaraldehydefixed or overfixed tissue is generally higher than for paraformaldehyde-fixed tissue. C aution: D eterm ine the optim al concentration of Proteinase K em pirically for each type of fixation. O ne of the m ost critical steps in the w hole procedure is finding the balance betw een prior fixation and the proper concentration of Proteinase K. Proteinase K concentrations w hich are too low or too high m ay lead to false negativ e results. (See Figure 3 under R esults.) O ur experience show s that autolysis causes few er problem s than ov erfixation does. Ev en after 30 hours autolysis, w e could dem onstrate m R N A . (See Figure 4 under R esults.) I n ov erfixed m aterial, w e w ere not alw ays successful in dem onstrating m R N A , ev en w hen w e increased Proteinase K to v ery high concentrations (up to 500 m g/ m l).
9 Rinse the sections 35 times with TBS. 10 Incubate the sections at 4C for 5 min
0.01% sheared salmon sperm DN A 0.02% SDS. 50% formamide.
(Sambrook et al., 1989).
C aution: T he concentration of dextran sulfate is critical. Without appropriate am ounts of dextran sulfate, the m ethod loses sensitiv ity. H ow ev er, excessiv e concentrations of dextran sulfate causes higher v iscosity of the hybridiz ation buffer. To prev ent unev en distribution of the probe and unev en signals, alw ays v ortex the hybridiz ation buffer extensiv ely.
3 Dilute the labeled antisense RN A probe
with TBS (pH 7.5) to stop the Proteinase K digestion. C aution: D o not postfix the sections w ith paraform aldehyde after protease digestion, since this reduces signal intensity.
11 Dehydrate the sections in a graded series
to an appropriate degree in hybridization buffer. The diluted probe solution may contain as much as 1 part labeled probe to 4 parts hybridization buffer. N ote: T he am ount of probe needed depends upon the degree of probe labeling (as determ ined in Procedure I , Step 4 abov e). G enerally, use the low est probe concentration that giv es optim al response in that dot blot procedure. Example: I n a serial dilution of the probe on a dot blot , if a 1:160 dilution giv es a significantly stronger response than a 1:320 dilution, perform the initial hybridiz ation experim ents w ith both a 1:200 and a 1:300 dilution of the probe.
4 Pipette the diluted
of ethanol solutions (increasing concentrations of ethanol). 12 Rinse the sections briefly with chloroform. N ote: A t this point, you m ay, if necessary, store the sections under dust- and R N ase-free conditions for sev eral days.
antisense probe solution onto each section at a volume of 10 l/cm 2. Cover with a coverslip. N ote: I n our experience, a prehybridiz ation step (w ith hybridiz ation buffer alone) did not im prov e results. same way whereby in steps 3 and 4 a labeled sense RN A probe is used. C aution: C ontrol hybridiz ation w ith sense probes is necessary to prov e the specificity of the reaction. H ow ev er, hybridiz ation w ith sense probes som etim es giv es unexpected hybridiz ation signals or back ground staining.
5 As a control serve sections treated in the
IV . Hybridization
C aution: Perform the hybridiz ation step under R N ase-free conditions. Prepare all solutions and buffers for this step w ith D EPC -treated w ater (Sam brook et al., 1989). Bak e all glassw are for 4 h at 180 C . A ssum e that disposable plasticw are is R N ase-free.
1 Place the sections in a humid chamber at
2 Prepare a hybridization buffer by mixing
6 Place the slides on a hot plate at 95C for
55C for 30 min.
the following components, then vortexing vigorously: 2 x SSC (Sambrook et al., 1989). 10% dextran sulfate.
4 min. N ote: T his step increases the signal from R N A / R N A hybrids. 7 Incubate the slides in a humid chamber for 46 h at 5575C. N ote: H ybridiz ation specificity can be increased by increasing the tem perature of hybridiz ation, if necessary, as high as 75 C . I ncreasing the tem perature can help to differentiate m R N A s of highly hom ologous proteins (Taylor et al., 1994). 137
CONTENTS
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V .W ashes and detection of mRNA

N ote: Stringency w ashings, often described as a m ethod to rem ov e nonspecifically bound probes, are helpful in reducing diffuse back ground staining, but do not significantly im prov e the specificity of the hybridiz ation.
1 Incubate the slides in 2 x SSC overnight.
1 Incubate slide overnight at 4C with
primary antibody (either polyclonal or mouse monoclonal), diluted as necessary in TBS containing 10% fetal calf serum (TBS-FCS). Example: T he polyclonal antibody for proteolipid protein used in Figure 1 w as diluted 1:1000.
2 Rinse the slide 35 times with TBS at
N ote: T he cov erslips w ill float off during this incubation.

2 Wash the slides as follows: 3 x 20 min at 55C with 50% deionized
room temperature. N ote: Perform Step 2 as w ell as all the rem aining incubation and w ash steps of Procedure V I at room tem perature.
3 Depending upon the type of primary
2 x15 min at room temperature with
formamide in 1x SSC.
1x SSC . 3 Rinse the sections 35 times with TBS. 4 Incubate the slides for 15 min with blocking mixture (blocking reagent containing 10% fetal calf serum). 5 Incubate the slides for 60 min with alkaline-phosphatase-conjugated antiDIG antibody [diluted 1: 500 in blocking mixture (from Step 4 above)]. 6 Rinse the sections 35 times with TBS. 7 Prepare N BT/BCIP color reagent as recommended by the manufacturer. 8 In a Coplin jar in the refrigerator, incubate the sections with color reagent until sufficient color develops. N ote: I ncubation can be extended up to 120 h if the color reagent is replaced ev ery tim e it precipitates or changes color.
9 Stop the color reaction by rinsing the
10 Finally, rinse the slides with distilled 11 Mount the slides directly with any water
antibody used in Step 1, do one of the following: If the primary antibody was a polyclonal antibody from rabbit, incubate the slide for 60 min with anti-rabbit serum from mouse [Dakopatts], diluted 1:100 in TBS-FCS, then rinse the slide 35 times with TBS. Go to Step 4. If the primary antibody was a mouse monoclonal antibody, skip this step and go to Step 4.
4 Incubate slide for 60 min with anti-mouse
slides several times with tap water. water.
soluble mounting medium. N ote: O ptionally, counterstain the slides or use im m unocytochem istry (as in Procedure V I below ) to v isualiz e proteins on the slides.
serum (from rabbit [Dakopatts], diluted 1:100 in TBS-FCS), then rinse the slide 35 times with TBS. 5 Incubate slide for 60 min with APAAP complex (mouse) (diluted 1:100 in TBSFCS), then rinse the slide 35 times with TBS. 6 Incubate slide for 30 min with anti-mouse serum (from rabbit) (diluted 1:100 in TBS-FCS), then rinse the slide 35 times with TBS. 7 Incubate slide for 30 min with APAAP complex (mouse) (diluted 1:100 in TBSFCS), then rinse the slide 35 times with TBS. 8 Repeat Steps 6 and 7. N ote: R epeating the A PA A P steps w ill enhance the signal.
9 Prepare APAAP substrate by dissolving
VI. Detection of proteins by immunocytochemistry

N ote: T his protocol describes a triple A PA A P (alk aline phosphatase anti-alk aline phosphatase) reaction (Vass et al., 1989), w hich allow s v isualiz ation of proteins on the sam e slide w ith the m R N A . See Figure 1 under R esults for an exam ple of double staining obtained w ith this technique.
200 mg naphthol-ASMX-phosphate, 20 ml dimethylformamide, and 1 ml of 1 M levamisole (all from Sigma) in 980 ml Tris-H Cl (pH 8.2). 10 Dissolve 50 mg Fast Red TR Salt in 50 ml of APAAP substrate, then filter the solution. 11 In a Coplin jar at room temperature, incubate the slides with the Fast RedAPAAP substrate solution until the color develops.
138
CONTENTS
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N ote: I f you substitute PA P com plex for A PA A P com plex, m odify the abov e procedure as follow s:
Perform all rinses and all dilutions w ith For the color reaction, use filtered dia-
PFA
GA
PBS rather than T BS.
m inobenz idine reagent (50 m g diam inobenz idine dissolv ed in 100 m l PBS w hich contains 0.01% H 2O 2) For an exam ple of im m unostaining w ith PA P com plex, see Figure 2 under R esults.
0.002%
Results
0.005%
Figure 1: Double staining of mRNA and protein (APAAP method) in a normal rat brain. The mRNA (black-blue) for proteolipid protein (PLP) was detected by the procedure described in the text and stained with NBT/BCIP color reagent. Proteolipid protein (red) was detected with a 1:1000-diluted rabbit polyclonal antibody and visualized with APAAP and Fast Red TR Salt.
0.02%
0.05%
Figure 2: Double staining of mRNA and protein (PAP method) in a normal rat brain. The experiment is identical to that in Figure 1, except PLP (brown) was visualized with PAP and diaminobenzidine reagent.
Figure 3: Effect of paraformaldehyde and glutaraldehyde fixation on in situ hybridization of mRNA. PLP mRNA was detected in sections of rat spinal cord which had been fixed by different methods. Panel a: Sections were fixed with 4% paraformaldehyde and then treated with different concentrations of Proteinase K. From top to bottom the Proteinase Kconcentrations were: 0.002%, 0.005%, 0.02%, 0.05%. Panel b: Sections were fixed in 2.5% glutaraldehyde, then treated with the same concentrations of Proteinase K as in Panel a.
CONTENTS
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139
Figure 4: Effect of autolysis and overfixation on in situ hybridization of mRNA and immunocytochemical staining. In spite of pronounced autolytic changes that occur postmortem, a good PLP mRNA signal can be seen in human brain autopsy samples (Panel a). Although in well preserved and fixed experimental tissue (Panel b) both, the signal for in situ hybridization as a well as the signal for immunocytochemistry is much more distinct. Protein (red) and mRNA (blue-black) were stained as in Figure 1.
References
Breitschopf, H; Suchanek, G ;G ould, R. M.; Colman, D. R.; Lassmann, H. (1992) In situ hybridization with digoxigenin-labeled probes: Sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol. 84, 581587. Hayashi, S; G illam, I. C.; Delaney, A. D.; Tener, G . M. (1978) Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with 125I-labeled RNA. J. Histochem. Cytochem. 26, 677679. Sambrook, J; Fritsch, E. F .; Maniatis, T . (1989) Molecular cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, N.Y .: Cold Spring Harbor Laboratory Press.
Acknowledgments
The rabbit polyclonal antibody against proteolipid protein (PLP) was a gift from Dr. S. Piddlesden, University of Cardiff, UK.
Taylor, V .; Miescher, G . C.; Pfarr, S.; Honegger, P .; Breitschopf, H.; Lassmann, H.; Steck, A. J. (1994) Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain. Neuroscience 63, 163178. Vass, K.; Berger, M. L.; Nowak, T . S. Jr.; W elch, W . J.; Lassmann, H. (1989) Induction of stress protein HSP 70 in nerve cells after status epilepticus in the rat. Neurosci. Lett. 100, 259264.

Reagent Description For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase. Lyophilizate Cat. No. 1175 025 Pack size 1 Kit (2x10 labeling reactions) 25 mg 100 mg 500 mg 1g 100 g 500 g 1 kg 50 g 150 U (200 l) 8 ml
DIG RNA Labeling Kit* , ** , *** Proteinase K
161 519 745 723 1 000 144 1 092 766 127 434 708 968 708 976 1 096 176 1 093 274 1 681 451
Tris
Powder
Blocking Reagent Anti-DIG-AP NBT/BCIP
Powder Fab Fragments from sheep Stock solution
140
CONTENTS
INDEX
RNA-RNA in situ hybridization using DIG -labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction
M arc D eBlock and D irk D ebrouw er, Plant G enetic System s N .V., G ent, Belgium . N ote: T he follow ing article is reprinted w ith the perm ission of A cadem ic Press (C opyright 1993 by A cadem ic Press, I nc.) and H arcourt Brace & C om pany and is based on the original article: D eBlock , M ., D ebrouw er, D . (1993) R N A -R N A in situ hybridiz ation using digoxigenin-labeled probes: the use of high m olecular w eight polyv inyl alcohol in the alk aline phosphatase indoxyl-nitroblue tetrazolium. Analytical Biochemistry 216; 8889. The indoxyl-nitroblue tetrazolium (BCIPN BT) reaction is relatively slow. During the reaction, intermediates (indoxyls) diffuse away into the medium, making it difficult to localize the site of hybridization (Van N oorden and Jonges, 1987) and reducing the hybridization signal. In this paper, an improved nonradioactive RN A-RN A in situ hybridization protocol using alkaline phosphatase-conjugated digoxigenin- (DIG-) labeled probes is presented. The addition of polyvinyl alcohol (PVA) of high molecular weight (40100 kD) to the BCIP-N BT detection system enhances the alkaline phosphatase reaction and prevents diffusion of reaction intermediates, resulting in a twentyfold increase in sensitivity without increasing the background. Due to the more localized precipitation of the formazan, the site of hybridization can be determined more precisely.
I. Fixation, dehydration, and embedding

Follow the protocol described by Jackson (1992) to fix, dehydrate, and embed the tissue in paraffin, with the following modifications:
1 For fixation, prepare either of the follow-
ing solutions: 100 mM phosphate buffer, pH 7, containing 0.25% gluteraldehyde and 4% freshly depolymerized paraformaldehyde. Formalin-acetic acid (50% ethanol; 10% formalin, containing 37% formaldehyde; 5% acetic acid). 2 Using either of the solutions from Step 1, fix the tissue for 4 h at room temperature. During the fixation: Vacuum infiltrate the tissue (with a water aspirator) for 10 min once an hour. After each vacuum infiltration, renew the fixative solution. 3 Depending on the fixative used in Step 2, wash the fixed tissue as follows: If gluteraldehyde-paraformaldehyde was the fixative, wash the fixed tissue 2 x 30 min with 100 mM phosphate buffer, pH 7. If formalin-acetic acid was the fixative, wash the fixed tissue 2 x 30 min with 50% ethanol. 4 Dehydrate the tissue by incubating in the following series of ethanol solutions: Either 90 min (after gluteraldehydeparaformaldehyde fixation) or 30 min (after formalin-acetic acid fixation) at room temperature in 50% ethanol. 90 min at room temperature in 70% ethanol. O vernight at 4C in 85% ethanol. 3 x 90 min at room temperature in 100% ethanol.
CONTENTS
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141
Polymer PVP
Molecular weight 15 kD 25 kD 44 kD 6 kD 20 kD 10 kD 40 kD 70100 kD
Concentration b 10, 20, 30% 10, 20, 30% 10, 20, 30% 10, 20, 30% 10, 20, 30% 10, 20% c 10% d 10% d
Effect on the alkaline phosphatase BCIP-N BT reaction e Autoreduction of N BT
PEG PVA
N o enhancement (0.01 mM formazan) Approx. 4-fold enhancement (0.04 mM formazan) Approximately 6- to 8-fold enhancement (0.06 to 0.08 mM formazan)
Table 1: Influence of polymers on the alkaline phosphatase BCIP-NBT reactiona. a Each reaction was done with a total volume of 2 ml BCIP-NBT reaction mixture in a test tube. The BCIP-NBT reaction mixture was as described in the text, except that it contained 7.5 X 10 3 units alkaline phosphatase/ml and the polymer indicated in the table. Incubation was for 30 min at 24 C. b The polymers were dissolved in the alkaline phosphatase reaction buffer in concentrations between 10% and 30%. The concentrations are given as a percentage of weight to volume. c A solution of 30% 10 kD PV A was too viscous. d Solutions of 20 or 30% 40 kD PV A and 20 or 30% 70100 kD PV A were too viscous. e The amount of formazan formed was measured at 605 nm (Eadie et al., 1970). The enhancement factor is expressed with respect to the controls (no polymer added). If no polymer was added, about 0.01 mM formazan was formed. a b c
Figure 1: The influence of PVA on the alkaline phosphatase BCIP-NBT reaction in in situ hybridizations. The hybridizations were carried out on 10 m paraffin sections of stigmas and styles of tobacco with antisense and sense DIG -labeled RNA probes of pMG 07 (de S. G oldman et al., 1992). C, cortex tissue; TT , transmitting tissue; V , vascular tissue. Bars = 200 m. Panel a: Hybridization with antisense probe. T wenty percent 10 kDPV A was added to the alkaline phosphatase reaction buffer. Development was done for 4 h. Panel b: Hybridization with antisense probe. Ten percent 70100 kD PV A was added to the alkaline phosphatase reaction buffer. Development was done for 2 h. Panel c: Hybridization with sense RNA probe. Ten percent 70100 kD PV A was added to the alkaline phosphatase reaction buffer. Development was done for 20 h.
144
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References
DeBlock, M.; Debrouwer, D. (1993) RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high molecular weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. Anal. Biochem. 215, 8689. de S. G oldman, M. H.; Pezzotti, M.; Seurinck, J.; Mariani, C. (1992) Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins. Plant Cell 4, 10411051. Eadie, M. J.; Tyrer, J. H.; Kukums, J. R.; Hooper, W . D. (1970) Histochemie 21, 170180. Jackson, D. (1991) In situ hybridization in plants. In: G urr, S. J.; McPherson; Bowles, D. J. (Eds.) Molecular Plant Pathology, A Practical Approach. Oxford, England: Oxford University Press, Vol. 1, pp. 163174. Van Noorden, C. J. F .; Jonges, G . N. (1987) Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyltetranitro BT method. Histochem. J. 19, 94102.

Reagent DIG RNA Labeling Kit* , ** , *** Proteinase K Description For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase. Lyophilizate Cat. No. 1175 025 Pack size 1 Kit (2x10 labeling reactions) 25 mg 100 mg 500 mg 1g 100 mg 500 mg 1g 2g 10 g 25 g 50 g 100 g 25 mg 100 mg 2,000 units 10,000 units 1 Kit
161 519 745 723 1 000 144 1 092 766 109 495 109 509 223 646 197 777 708 984 1 583 786 708 992 709 000 109 142 109 169 799 017 799 025 1175 041
tRNA DNA from herring sperm DTT
From bakers yeast, lyophilizate Lyophilized, sodium salt Purity: > 97%
RNase A RNase Inhibitor DIG Nucleic Acid Detection Kit*
Powder From human placenta For detection of digoxigenin-labeled nucleic acids by an enzyme-linked immunoassay with a highly specific antiDIG -AP antibody conjugate and the color substrates NBT and BCIP .
CONTENTS
INDEX
145
II. Sectioning
1 Cut the paraffin-embedded tissues in
Incubate 416 h at 20C. Centrifuge in a microcentrifuge for 15 Discard the supernatant and dry the Resuspend labeled probe RN A in
4 Prepare a hybridization mixture con
min at 4C to pellet the RN A.
10 m sections. 2 Attach the sections at 75C for 1 h to slides that have been treated with Vectabond (Vector Laboratories, Burlingame, CA, U.S.A.).
RN A pellet in a vacuum desiccator.
DEPC-treated water at 1050 g/ml.
III. Prehybridization treatments

1 Dewax and hydrate the sections as
2 Incubate the sections with a Proteinase K
previously described (Jackson, 1992).
solution (100 mM Tris, pH 7.5; 50 mM EDTA; 2 g/ml Proteinase K) for 30 min at 37C. 3 After the Proteinase K treatment, wash the slides 2 x in phosphate buffered saline (PBS). 4 Dehydrate the sections in ascending concentrations of ethanol as previously described (Jackson, 1991).
taining the following: 50% deionized formamide. 2.25 x SSPE (300 mM N aCl; 20 mM N aH 2PO 4; 2 mM EDTA; pH 7.4). 10% dextran sulfate. 2.5 x Denhardts solution. 100 g/ml sheared and denatured herring sperm DN A. 100 g/ml tRN A. 5 mM DTT. 40 units/ml RN ase inhibitor. 0.21.0 g/ml hydrolyzed and denatured probe (200 bases long).
5 Cover each section with 250500 l of
IV . Hybridization
1 Label the probe RN A with DIG-UTP
hybridization mixture (depending on the size of the section) and incubate in a humidified box at 42C overnight. N ote: D o not use a cov erslip during the hybridiz ation incubation.
6 After hybridization, wash the slides as
according to the procedures given in Chapter 4 of this manual. 2 Reduce the length of the probe to approximately 200 bases as follows: To 50 l of labeled probe RN A in a microcentrifuge tube, add 30 l 200 mM N a2CO 3 and 20 l 200 mM N aH CO 3. H ydrolyze the probe at 60C for t min, where: t = (L 0 L f) / (K L 0 L f) L 0 = starting length of probe RN A (in kb) L f = length of probe RN A (in kb) (In this case, L f = 0.2 kb.) K = rate constant (In this case, K = 0.11 kb/min.) t = hydrolysis time in min
3 After hydrolysis, purify the probe RN A
follows: 5 min at room temperature with 3 x SSC. N ote: 1 x SSC contains 150 m M N aC l, 15 m M N a-citrate; pH 7.
5 min at room temperature with N TE
as follows: Add the following to the hydrolyzed probe solution: 5 l 10% acetic acid 11 l 3 M sodium acetate (pH 6.0) 1 l of a 10 mg/ml tRN A stock 1.2 l 1 M MgCl2 300 l (about 2.5 volumes) cold ethanol
(500 mM N aCl, 10 mM Tris-H Cl, 1 mM EDTA; pH 7.5). 7 To remove unhybridized single-stranded RN A probe, put slides into a humidified box and cover each section with 500 l of N TE buffer containing 50 g/ ml RN ase A. Incubate for 30 min at 37C. 8 After RN ase treatment, wash the slides 3 x 5 min at room temperature with N TE. 9 To remove nonspecifically hybridized probe, wash the slides as follows: 30 min at room temperature with 2 x SSC. 1 h at 57C with 0.1 x SSC.
142
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V . Detection of DIG -labeled hybrids

1 Incubate the slides first with blocking
6 Dehydrate the sections by incubating the
solution, then with blocking solution containing 1.25 units/ml of alkaline phosphatase-conjugated anti-DIG Fab fragments as recommended in the pack insert of the DIG N ucleic Acid Detection Kit*. 2 After the antibody incubation, wash the slides to remove unbound antibody as recommended in the Boehringer Mannheim DIG/Genius System Users Guide for Membrane H ybridization. 3 Prepare the BCIP-N BT-PVA color development solution as follows: Prepare a Tris-N aCl-PVA stock solution by dissolving 10% (w/v) polyvinyl alcohol [PVA, either 40 kD or 70100 kD (Sigma)] at 90C in 100 mM Tris-H Cl (pH 9) containing 100 mM N aCl. Cool the Tris-N aCl-PVA stock solution to room temperature. Add MgCl2, BCIP, and N BT to the Tris-N aCl-PVA stock to produce a final color development solution that contains: 5 mM MgCl 0.2 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP) 0.2 mM nitroblue tetrazolium salt (N BT). 4 After the washes in Step 2, perform the visualization step as follows: Place the slides in 30 ml of BCIPN BT-PVA color development solution in a vertical staining dish suited for eight slides. Incubate the slides in the color development solution for 216 h at 30C. Monitor color formation visually. 5 When the color on each slide is optimal, stop the color reaction by washing the slide 3 x 5 min in distilled water.
slides without shaking in the following ethanol solutions: 15 s in 70% ethanol. 2 x15 s in 100% ethanol. 7 Air dry the slides and mount them with Eukitt (O . Kindler GmbH , FRG). 8 Examine the sections with a Dialux 22 microscope (Leitz, Wetzlar, FRG) equipped with N ormaski differential interference contrast.
Results and discussion

The direct influence of the polymers on the BCIP-N BT alkaline phosphatase reaction in a test tube is shown in Table 1. From these results it is clear that polyvinyl alcohol was the only polymer that enhanced formazan formation in the alkaline phosphatase reaction. This enhancement was even more pronounced in in situ hybridization experiments. Figure 1 shows the results of an in situ hybridization in which sections of tobacco pistils were hybridized with an antisense RN A probe from a pistil-specific cDN A clone (de S. Goldman et al., 1992). After three hours development no hybridization could be detected if no PVA had been added to the reaction buffer. A clear but still weak signal was visible after three h if 20% PVA (molecular weight, 10 kD) was used (Figure 1a). H owever, with 10% PVA (molecular weight, 70100 kD), a strong hybridization signal appeared in the transmitting tissue of the pistil after a few h (Figure 1b). In the control with the sense RN A probe no signal could be detected, even after 24 h of development (Figure 1c).
CONTENTS
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143
Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG -dUTP
D epartm ent of C ytochem istry and C ytom etry, U niv ersity of L eiden, T he N etherlands. The protocol given below has been developed with the neuropeptidergic system of the pond snail Lymnaea stagnalis. More information concerning the application and in situ hybridization methodology can be found in Van Minnen et al. (1989), Dirks et al. (1988), Dirks et al. (1990), and Dirks et al. (1991).
1 Perform the hybridization as follows: Prepare hybridization mixture [25%
formamide; 3 x SSC; 0.1% polyvinylpyrrolidone; 0.1% ficoll, 1% bovine serum albumin; 500 g/ml sheared salmon sperm DN A; 500 g/ml yeast RN A]. Note: 1x SSC contains 150 mM sodium chloride, 15 mM sodium citrate; pH 7.0.
To the cryostat sections, add hybridi-
I. Tissue preparation
1 Dissect the tissue, embed in O .C.T.
compound (Miles Scientific, USA), and freeze in liquid nitrogen. 2 Prepare sections as follows: Cut cryostat sections of 8 m. Mount on poly-L-lysine-coated slides. Air dry. 3 After mounting, do the following: Fix the sections for 30 min at 4C with modified Carnoys [2% formalin, (from 37% stock), 75% ethanol, 23% acetic acid]. Rinse the slides with water. Dehydrate the sections.
zation mixture containing 1 ng/l labeled probe. Incubate for 2 h at room temperature. N ote: I f the probe contains a fluorochrom e label, perform the hybridiz ation incubation in the dark .
2 After hybridization, do the following: Rinse the section 3 times in 4 x SSC at
Dehydrate the sections.
room temperature.
N ote: I f the probe contains a fluorochrom e label, perform the w ashes in the dark .
IV . Hybrid detection II. Probe preparation

Synthesize and purify the oligonucleotides according to routine procedures. Tail the oligonucleotide with DIG-dUTP or fluorescein-dUTP according to the procedures given in Chapter 4, but without using dATP. N ote: I f you use fluorescein-dU T P, add to the labeling m ixture equal am ounts of unm odified dT T P and fluorescein-dU T P, then carry out the labeling procedure in the dark . Depending on the label used on the probe, follow one of these procedures: If sections are hybridized with fluoresceinlabeled oligonucleotides: Mount sections in PBS/glycerol (1:9; v/v) containing 2.3% 1,4-diazabicyclo(2,2,2)-octane (DABCO , from Sigma) and 0.1 g/l 4',6'-diamidino-2phenylindole (DAPI). Evaluate under a fluorescence microscope. If sections are hybridized with DIGlabeled oligonucleotides: Make a 1:250 dilution of fluoresceinconjugated anti-DIG antibody (from sheep) in incubation buffer [100 mM Tris-H Cl, pH 7.4; 150 mM N aCl; 1% blocking reagent]. O verlay the sections with the diluted antibody in incubation buffer. Incubate sections at room temperature for 30 min.
III. In situ hybridization

N ote: 18-m ers are used in this study. For oligonucleotides w ith other lengths and/ or com position, you m ay hav e to alter the stringency of hybridiz ation by changing the form am ide concentration or the hybridiz ation tem perature listed below.
146
CONTENTS
INDEX
Rinse sections 3 x 5 min in Tris-N aCl Mount and evaluate as for fluorescein-
[100 mM Tris, pH 7.4; 150 mM N aCl]. labeled oligonucleotides.
Results
Figure 2: Indirect detection of
caudodorsal cells with DIG-labeled CDCH-I and sheep anti-DIG-fluorescein conjugate. The same cells are positive, but the hybridization signal is clearly more intense than that obtained with the direct technique (Figure 1).
Figure 1: Direct detection of caudodorsal cells with fluorescein-labeled CDCH-I.
References
Dirks, R. W .; van G ijlswijk, R. P . M.; Vooijs, M. A.; Smit, A. B.; Bogerd, J.; Van Minnen, J.; Raap, A. K.; Van der Ploeg, M. (1991) 3'-end fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple, simultaneous RNA detection. Exp. Cell Res. 194, 310315. Dirks, R. W .; van G ijlswijk, R. P . M.; Tullis, R. H.; Smit, A. B.; Van Minnen, J.; Van der Ploeg, M.; Raap, A. K. (1990) Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITCand biotin-labeled oligonucleotides. J. Histochem. Cytochem. 38, 467473. Dirks, R. W .; Raap, A. K.; Van Minnen, J.; Vreugdenhil, E.; Smit, A. B.; Van der Ploeg, M. (1989) Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaeae stagnalis by radioactive and nonradioactive in situ hybridization: a model study for mRNA detection. J. Histochem. Cytochem. 37, 714. Van Minnen, J.; Van de Haar, Ch.; Raap, A. K.; Vreugdenhil, E. (1988) Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaeae stagnalis by means of immunocytochemistry and in situ hybridization. Cell Tissue Res. 251, 477484.

Reagent DIG Oligonucleotide Tailing Kit* , ** , *** Fluorescein-12-dUTP tRNA Anti-DigoxigeninFluorescein Anti-DigoxigeninRhodamine DAPI Blocking Reagent BSA Description For tailing oligonucleotides with digoxigenin-dUTP . Tetralithium salt, solution From bakers yeast, lyophilizate Fab Fragments from sheep Fab Fragments from sheep Fluorescence dye for staining of chromosomes Blocking reagent for nucleic acid hybridization Highest quality, lyophilizate Cat. No. 1 417 231 1 373 242 109 495 109 509 1 207 741 1 207 750 236 276 1 096 176 238 031 238 040 Pack size 1 Kit (25 tailing reactions) 25 nmol (25 l) 100 mg 500 mg 200 g 200 g 10 mg 50 g 1g 10 g
***Sold under the tradename of G enius in the US. ***EP Patents 0 124 657/0 324 474 granted to Boehringer Mannheim G mbH. ***Licensed by Institute Pasteur.
CONTENTS
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In situ hybridization of DIG -labeled rRNA probes to mouse liver ultrathin sections
D r. D . Fischer, D . Weisenberger, and Prof. D r. U . Scheer, I nstitute for Z oology I , U niv ersity of W rz burg, G erm any.
4 Transfer the liver cubes to other petri
Thin section electron microscopy of nucleoli from mammalian cells reveals three nucleolar substructures. These are the fibrillar centers (FC), the dense fibrillar component, which forms a closely apposed layer upon the FCs, and the granular component, which constitutes the bulk of the nucleolar mass. Although there is no doubt that this ultrastructural organization somehow reflects the vectorial process of ribosome formation, the exact structurefunction relationships have been a matter of much discussion and are still largely unclear. (For a review, see Scheer and Benavente, 1990.) H ere we describe an approach to localize specific intermediates for the pre-rRN A maturation process using in situ hybridization at the ultrastructural level. This approach will allow the correlation of each pre-rRN A processing step with the defined nucleolar substructures. The procedure described can also be applied to the localization of other RN A species involved in ribosome biogenesis, such as 5S rRN A or U3 snRN A (Fischer et al., 1991).
dishes on ice, containing the fixatives (i) 4% PFA and (ii) 0.5% GA plus 4% PFA. 5 Incubate the cubes in the fixatives for about 2 h. 6 To remove the fixative, wash the cubes 3 x 5 min with ice cold PBS.
IB. Dehydration of material
Transfer the object cubes to preparative glasses and dehydrate them in a series of ethanol solutions according to the following scheme: 2 x15 min in 30% ethanol at 4C. 1x 30 min in 50% ethanol at 4C. 1x 30 min in 50% ethanol at 20C. 2 x 30 min in 70% ethanol at 20C. 2 x 30 min in 90% ethanol at 20C. 2 x 30 min in 96% ethanol at 20C. 2 x 30 min in 100% ethanol at 20C. C aution: L eav e the objects in a sm all am ount of liquid during changes from one ethanol solution to another. T his prev ents them from drying out, especially at higher ethanol concentrations.
IC. Infiltration
I. Embedding in Lowicryl K4M

(according to C arlem alm and V illiger, 1989, w ith alterations)
IA. Fixation of material
1 Prepare the following fixation solutions
After dehydration, ethanol must be infiltrated with the embedding medium Lowicryl K4M (Chemische Werke Lowi, Waldkraiburg, FRG). This resin tolerates a little residual water in the object and produces good results in immunocytochemistry. C aution: W hen w ork ing w ith L ow icryl K4M , perform all steps: At low temperatures Under the fume hood While wearing gloves While excluding air (oxygen) from K4M as much as possible. To exclude air from the embedding medium, do one of the following: Fill all vessels which contain K4M with nitrogen. Perform all steps in a closed chamber cooled to constant temperature by liquid nitrogen, thus providing a nitrogen-saturated atmosphere in the closed preparation chamber (e.g., CS-auto, Reichert & Jung, Cambridge Instr.).
and keep them on ice: 4% paraformaldehyde (PFA) in PBS (137 mM N aCl, 2.7 mM KCl, 7 mM N a2H PO 4, 1.5 mM KH 2PO 4). 0.5% glutaraldehyde (GA) plus 4% PFA in PBS. 2 Take a piece of freshly prepared mouse liver and submerge it in a petri dish filled with 4% PFA in PBS on ice. 3 Cut the liver into small cubes with a maximal side length of 1 mm. N ote: T he sm aller the pieces, the better reagent penetrates them in the procedure below.
148
CONTENTS
INDEX
1 Prepare Lowicryl K4M as follows (to
produce a medium hard plastic): Mix 2 g Crosslinker A, 13 g Monomer B, and 0.075 g Initiator C. Dissolve Initiator C by gentle stirring or by bubbling dry nitrogen through the mixture. Cool the mixture to 20C in the freezer. 2 Perform infiltration at about 20C with suitably precooled material according to the following scheme: 1h with 100% ethanol:K4M (1:1, v/v). 2 x1 h with K4M. O vernight with K4M. 2 x 3 h with K4M.
ID. Embedding
II. Sectioning
The embedded mouse liver cubes can easily be recognized in the transparent resin Lowicryl K4M. Carlemalm and Villiger (1989) recommend trimming with the ultramicrotome and a glass knife to get smooth surfaces.
1 O btain semithin sections with a glass
knife and ultrathin sections with a diamond knife, according to standard protocols. C aution: Because of the hydrophilicity of L ow icryl K4M , be careful not to w et the surface of the block .
2 Transfer sections to 200 mesh nickel grids
Gelatin capsules (e.g. Capsulae O perculatae N o. 2) are very suitable as molds for embedding because they are transparent to UV-light and can afterwards easily be cut away. 1 Cool the gelatin capsules to the appropriate temperature. 2 Add a few drops of Lowicryl K4M to each capsule. 3 Drop one object cube into each capsule. Caution: Do this transfer very carefully. Do not mechanically damage the cube, allow it to dry out, or allow it to warm up.
4 Fill the capsules with the embedding 5 Let the capsules stand for about half an
which have been coated with 0.5% parlodion. 3 Air dry the sections. N ote: N ow the sections are ready for hybridiz ation.
III. Probe preparation

1 Clone restriction fragments from mouse
2 Prepare in vitro transcripts from the
rDN A.
medium.
hour to allow the air bubbles to leave.

IE. Polymerization
1 Before switching on the UV light, turn
the temperature a bit lower to compensate for the heat from the light. 2 Induce polymerization of Lowicryl K4M by 360 nm long-wave UV radiation (CS-UV, Reichert & Jung, Cambridge Instr.) over a five day period. During the polymerization, do the following: For about three days, keep the polymerization temperature constant at any temperature between 0C and 40C. After three days, allow the temperature to rise to room temperature with about 8 K/h. N ote: For troubleshooting, see C arlem alm and V illiger (1989).
rDN A clones and label them with DIGUTP according to the protocols in Chapter 4 of this manual. 3 Since probe length might influence the efficiency of in situ hybridization experiments, monitor transcript length by electrophoresis (in formaldehyde-containing gels), blotting onto nitrocellulose, and detection with the anti-D IG -alkaline phosphatase conjugate. 4 If necessary, reduce probe length to approximately 150 nucleotides by limited alkaline hydrolysis of the transcripts according to Cox et al. (1984). Briefly, the procedure is: Incubate the transcripts with a solution of 40 mM N aH CO 3 and 60 mM N a2CO 3 at 60C. Calculate the Incubation time as follows: L0 Lf t = ________ k L0 Lf L 0 = initial length of transcript (in kb) L f = desired probe length (in kb) k = constant = 0.11 kb/min
Stop the hydrolysis by adding 3 M Precipitate the hydrolyzate with ethanol. Dissolve the precipitate in water.
sodium acetate and acetic acid.
CONTENTS
INDEX
149
IV . Hybridization
N ote: Perform all the follow ing steps (including the hybridiz ation) in a m oist cham ber.
1 Prepare hybridization mixture [5 x SSC;
3 Apply diluted antibody to section and

4 Wash the sections as follows: 3 x 5 min with PBST. 6 x 5 min with redistilled water. 5 Perform silver enhancement by incubating
incubate for 1 h.
0.1 mg/ml tRN A; DIG-labeled antisense transcript at a final concentration of 10 ng/l]. N ote: 1 x SSC contains 0.15 M N aC l, 0.015 M sodium citrate. 2 To monitor the specificity of binding, prepare a negative probe by substituting the sense transcript for the antisense transcript in the hybridization mixture above. 3 Place a droplet of the appropriate hybridization mixture onto Parafilm . 4 Incubate the ultrathin sections by putting the grid (section-side down) on top of the droplet. 5 Perform hybridization for at least 3 h at 65C. 6 Wash the sections as follows, all at room temperature: 3 x 5 min with 2 x SSC. 2 x10 min with PBST (PBS containing 0.1% Tween 20).
sectionswith developer and enhancer (from the set of silver enhancement reagents) at a ratio of 1:1 for 420 min (depending on the desired silver grain size). 6 Wash the sections 6 x 5 min with redis tilled water. 7 Stain with 2% aqueous uranyl acetate (4 min) and Reynolds lead citrate for 1 min (Reynolds, 1963) according to standard procedures. 8 Evaluate the specimen in the electron microscope.
Results
In Figure 1, the hybridized probe was detected with monoclonal anti-DIG antibody coupled to 1 nm gold particles. The signal was enhanced with silver staining for 6 min at room temperature. Silver intensification facilitates visualization of the bound hybridization probe at low magnification. The silver grains scattered throughout the nucleoplasm might indicate transport of preribosomal particles from the nucleolus to the cytoplasm. In Figure 2, a digoxigenin-labeled riboprobe complementary to the first 3 kb of the 5' region of the ETS1 was hybridized to a Lowicryl section of mouse liver and detected as described in Figure 1.
V . Detection
1 To block nonspecific sites, incubate each
section for 15 min with PBST plus BG (PBST containing 1% bovine serum albumin, 0.1% CWFS-gelatin). 2 Dilute 1 nm gold-conjugated anti-DIG antibody 1:30 in PBST plus BG.
Figure 1: In situ hybridization of a digoxigeninlabeled RNA probe complementary tomost of the 28 S rRNAregion toan ultrathin section of mouse liver fixed with 3% formaldehyde and embedded in Lowicryl K4M. The nucleolus as well as the cytoplasm are clearly marked. Label is essentially absent from the mitochondria, demonstrating the specificity of the method. Cytoplasmic labeling is due to labeling of the ribosomes, especially the endoplasmatic reticulum-associated ones (Magnification: 25,200x).
Figure 2: Localization of pre-rRNA molecules containing the 5' region of the ETS1. For details of the probe, see the text. The survey view shows selective labeling of the fibrillar components of the nucleolus (Magnification: 27,000x).
150
CONTENTS
INDEX
References
Carlemalm, E.; Villiger, W . (1989) Low temperature embedding. In: Bullock, G . R.; Petrusz, P . (Eds) Techniques Immunocytochem. 4, 2944. Cox, K. H.; DeLeon, D. V .; Angerer, L. M.; Angerer, R. C. (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101, 485502. Fischer, D.; W eisenberger, D.; Scheer, U. (1991) Assigning functions to nucleolar structures. Chromosoma 101, 133140. Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208212. Scheer, U.; Benavente, R. (1990) Functional and dynamic aspects of the mammalian nucleolus. BioEssays 12, 1421.

Reagent DIG RNA Labeling Kit* , ** , *** Tween 20 BSA Anti-DigoxigeninGold Silver Enhancement Reagents Highest quality, lyophilizate Affinity purified sheep IgG to digoxigenin conjugated with ultra small gold particles (average diameter ! 0.8 nm). Detection of colloidal gold particles (anti-DIG -gold) by silver deposition on the particle surface Description For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase. Cat. No. 1175 025 Pack size 1 Kit (2x10 labeling reactions) 5x10 ml 1g 10 g 1 ml
1 332 465 238 031 238 040 1 450 590
1 465 350
1 set
CONTENTS
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151
RNA in situ hybridization using DIG -labeled cRNA probes

H . B. P. M . D ijk m an, S. M entz el, A . S. de Jong, and K. J. M . A ssm ann D epartm ent of Pathology, N ijm egen U niv ersity H ospital, N ijm egen, T he N etherlands. In recent years, the in situ hybridization (ISH ) technique has found widespread application in both basic science and diagnostic clinical research. The ISH technique has frequently been used to localize specific genes on metaphase chromosomes and to detect viral and bacterial genomes in infected tissues. The RN A in situ hybridization (RISH ) technique for the examination of mRN A expression has gained less attention due to the many technical problems associated with this technique. In this report, we present a step-by-step protocol for a nonradioactive RISH technique on frozen sections using digoxigeninlabeled copy RN A (cRN A) probes. We demonstrate this technique on frozen sections of mouse kidney using DIG-labeled cRN A probes for the ectoenzyme aminopeptidase A, a low-copy RN A (Assmann et al., 1992). For a high-copy RN A, we studied human psoriatic epidermis using a DIGlabeled cRN A probe for elafin/SKALP, an inhibitor of leukocyte elastase and proteinase 3 (Alkemade et al., 1994). This protocol has been optimized to give strong hybridization signals, even with low-copy mRN A molecules. For a full discussion of each step of this protocol, along with more hints on enhancing the result of this often laborious and troublesome technique, see the previously published version of this report (Dijkman et al., 1995a; 1995b).
I. Probe selection
Choosing the type of probe (i.e., DN A, RN A, or oligonucleotide) is essential for a good final result. For the optimization of the probe labeling, we have tested four methods of incorporating the DIG label:
Direct labeling of cDN A molecules using DIG labeling of oligonucleotides using DIG labeling of PCR products according DIG labeling of cRN A molecules using
the DIG DN A Labeling Kit*.
the DIG O ligonucleotide Tailing Kit*. to the method of H annon et al. (1993). the DIG RN A Labeling Kit*.
The RN A labeling method gave by far the best results. Therefore, we provide some hints on how to transcribe DIG-labeled cRN A molecules:
For cRN A probe synthesis, subclone a
cDN A molecule in an appropriate vector. The vector should have sequences flanking the insert that allow the insert to be transcribed. Typically, commercially available vectors have SP6 and T7 RN A polymerase sites flanking the multiple cloning sites. Regulate the length of the cDN A molecule since this greatly affects the hybridization efficiency. Usually, a cDN A length between 200 and 500 nucleotides gives the best results, allowing efficient hybridization and good penetration of the tissue. When a molecule of interest is expressed as a high-copy RN A but the target RN A is masked by proteins, make probes between 100 and 200 nucleotides long for better penetration (Figure 1).
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II. Probe labeling

1 After subcloning the cDN A molecule,
linearize the circular vector with restriction enzymes that cut the multiple cloning site in 2 orientations, allowing sense and antisense synthesis. C aution: C ontrol this lineariz ation step carefully by m onitoring the digestion w ith agarose gel electrophoresis. C ircular m olecules that are left in the digestion m ixture affect the transcription efficiency.
2 Extract the linearized molecules from the
3 Label the transcripts from the linearized

b
digestion mixture by phenol extraction.
molecules with DIG-labeled nucleotides and the DIG RN A Labeling Kit* according to the instructions given in the kit, but with the following modifications: Perform the SP6 polymerase incubation at 40C instead of 37C. Precipitate the labeled molecules overnight at 20C, rather than 30 min at 70C. Monitor the transcription reaction by agarose gel electrophoresis to check for correct cRN A probe length. 4 Monitor probe labeling by spotting diluted aliquots of the labeled cRN A probes on nylon membranes and analyzing with the DIG Luminescent Detection Kit*. See also Chapter 4, page 51. N ote: T he sense and antisense cR N A probes should be labeled equally as efficiently as the control D N A included in the D I G R N A L abeling Kit*.
5 If necessary, adjust the concentration of
the labeled sense and antisense cRN A probes so they contain equal amounts of label. 6 Aliquot the cRN A probes in poly propylene tubes at 70C. N ote: R epeated freez ing and thaw ing affects the labeled cR N A probe.
Figure 1: RNA in situ hybridization on a frozen section of human psoriatic epidermis with DIGlabeled cRNA probes coding for human SKALP . Panel a: An antisense cRNA probe (150 bp) was used on a 10 m thick section (magnificationx125). Panel b: An antisense cRNA probe (150 bp) was used on a 20 m thick section (magnificationx500). Panel c: A sense cRNA probe (150 bp) was used on a 10 m thick section (magnificationx270). Intense staining of both the stratum spinosum and stratum granulosum is observed with the antisense probe. *Sold under the tradename of G enius in the US.
CONTENTS
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153
III. Preparation of tissue sections

N ote: For detection of low -copy R N A m olecules, alw ays prepare froz en sections, since paraffin em bedding causes a loss of approxim ately 30% of the R N A . T he m ethod described here has been optim iz ed for froz en sections.
1 For the processing of tissue, clean all
IV . Pretreatment of slides
C aution: Prepare all solutions for Procedures I V w ith w ater that has been treated w ith 0.1% D EPC .
1 Directly after mounting, heat the sections
knives and other materials with RN ase ZAP (AMBIO N ) and work as aseptically as possible. 2 Remove the tissue from the animal, immediately snap-freeze the tissue, and store it in liquid nitrogen. Work quickly to avoid degradation of RN A (Barton et al., 1993). 3 Cut 10 m thick frozen sections. N ote: To get a higher signal, cut sections thick er than 10 m . For better signal localiz ation, cut sections thinner than 10 m .
4 Mount tissue directly on Superfrost Plus
slides (Menzel Glser, O mnilabo, Breda, The N etherlands) to prevent detachment of the section during the RISH procedure.
on a stove for 2 min at 50C to fix the RN A in the tissue. N ote: Vary the tim e (from 10120 s) and tem perature (from 5090 C ) of the fixation step to accom m odate different types of tissue and R N A . 2 Dry the sections for 30 min. 3 Circle the sections with a silicone pen (DAKO A/S, Glostrup, Denmark) to prevent smudging of the substrate. 4 Use the following criteria to decide the next step of the procedure: If the target tissue has lipid vesicles (Figure 2) that interfere with the RISH detection, go to Step 5. If the target tissue does not have lipid vesicles that interfere with the RISH detection, go to Step 6.
Figure 2: RNA in situ hybridization on a frozen kidney section with a DIG-labeled cRNAprobe for mouse aminopeptidase A(without delipidization). The section was 10 M thick and was taken from a male BALB/c mouse. Note the many nonspecific lipid vesicles in the section. The specific hybridization signal appears in cells of the glomerulus (arrows). Magnification, 600 x.
5 (O ptional)
To minimize nonspecific background caused by lipid vesicles, do the following (all at room temperature): Delipidize the sections by extracting them for 5 min in chloroform. Dry the section to evaporate the chloroform. N ote: T his delipidation step m ay be om itted in pilot studies on a new tissue. 6 Fix the tissue sections as follows (all at room temperature): Incubate tissue in PBS containing 4% paraformaldehyde for 7 min. Wash 1 x 3 min with PBS. Wash 2 x 5 min with 2 x SSC. N ote: 1 x SSC contains 150 m M N aC l, 15 m M sodium citrate; pH 7.2. 154
CONTENTS
!
INDEX
V . Prehybridization, hybridization, and posthybridization

C aution: Prepare all solutions for Procedures V w ith w ater that has been treated w ith 0.1% D EPC .
1 Prehybridize each section for 60 min at
VI. Immunological detection

1 Wash the sections for 5 min at room
37C in 100 l hybridization buffer [4 x SSC ; 10% dextran sulfate; 1 x D enhardts solution (0.02% Ficoll 400, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin); 2 mM ED TA; 50% deionized formamide; 500 g/ ml herring sperm D N A]. N ote: You m ay v ary the tem perature of the prehybridiz ation and hybridiz ation steps betw een 37 C and 50 C .
2 Perform hybridization as follows: Remove and discard the buffer from
Cover each section with 100 l hybridi-
the prehybridization step.
zation buffer containing 200 ng/ml of DIG-labeled cRN A probe. C aution: D o not use cov erslips, since they decrease the signal up to fourfold. C aution: A probe concentration of 200 ng/ m l holds only if the cR N A w as labeled efficiently. I f, in Procedure I I , the cR N A probe w as not labeled as efficiently as the controls from the D I G R N A L abeling Kit*, adjust the concentration of the cR N A probe in the hybridiz ation m ixture.
Incubate for 16 h at 37C.
temperature with 100 mM Tris-H Cl (pH 7.5), 150 mM N aCl. 2 Incubate the sections for 30 min at room temperature with blocking buffer [100 mM Tris-H Cl (pH 7.5), 150 mM N aCl; saturated with blocking reagent]. 3 Prepare a 1:200 dilution of alkaline phosphatase-conjugated anti-DIG antibody (polyclonal, Fab fragments, from sheep) in blocking buffer (from Step 2). 4 Incubate the sections for 120 min at room temperature with the diluted anti-DIG antibody conjugate prepared in Step 3. 5 Wash the sections as follows: 2 x 5 min at room temperature with 100 mM Tris-H Cl (pH 7.5), 150 mM N aCl. 1x10 min at room temperature with 100 mM detection buffer [Tris-H Cl (pH 9.5), 100 mM N aCl, 50 mM MgCl2]. 6 Cover the sections with detection buffer containing 0.18 mg/ml BCIP, 0.34 mg/ml N BT, and 240 g/ml levamisole. Incubate for 16 h at room temperature (for detection of low abundance RN A). N ote: T his dev elopm ent should be carried out w ith the slides standing up in a sm all container to prev ent nonspecifically conv erted substrate from falling onto the section.
7 Stop the color reaction by washing the
N ote: You m ay v ary the tem perature of the prehybridiz ation and hybridiz ation steps betw een 37 C and 50 C .
3 After the hybridization, wash unbound
sections for 5 min in 10 mM Tris (pH 8), 1 mM EDTA.
cRN A probe from the section as follows: 1 x 5 min with 2 x SSC at 37C. N ote: To m ak e the w ash m ore stringent and w ash aw ay nonspecifically bound cR N A probe, low er the salt concentration or increase the form am ide concentration in the w ashing buffer and perform the w ashing step at a tem perature 5C beneath the m elting tem perature of the probe.
3 x 5 min with 60% formamide in 2 x 5 min with 2 x SSC at room tem-
VII. Counterstain
1 Wash the sections for 5 min with distilled
2 Counterstain the sections for 510 min
H 2O at room temperature.
3 Repeat wash (from Step 1). 4 Mount coverslips in Kaisers solution
with 1% methylene green at 37C.
(available from Merck).
0.2 x SSC at 37C. perature.
CONTENTS
INDEX
155

Figure 3 shows a typical RISH signal obtained with this protocol and a labeled antisense cRN A probe coding for mouse aminopeptidase A. The protocol included the optional delipidation step with chloroform (Procedure IV, Step 5). Figure 4 shows the control reaction with the labeled sense cRN A probe. We have provided a standard protocol for RISH on low- and high-copy RN A molecules. Using this protocol, one should be able to perform RISH on each RN A molecule of interest, with only minor modifications depending on the abundance of the RN A of interest.
Acknowledgments
The cDN A clone coding for the mouse aminopeptidase A cDN A sequence was kindly provided by Dr. Max D. Cooper from the University of Alabama at Birmingham, USA (Wu et al., 1990). The cRN A probe for elafin/SKALP was kindly provided by Dr. J. Schalkwijk, department of Dermatology, University H ospital N ijmegen, The N etherlands.
Figure 3: RNA in situ hybridization on a frozen kidney section with a DIG-labeled antisense cRNA probe for mouse aminopeptidase A (after delipidization). The section was 10 m thick and was taken from a male BALB/c mouse. Pretreatment of the section with chloroform prior to the hybridization delipidized the section and reduced a nonspecific signal previously observed in lipid vesicles (Figure 2). The specific hybridization signal (arrows) was undiminished by the chloroform
Figure 4: RNA in situ hybridization on a frozen kidney section from the same mouse used in Figure 3 with a DIG-labeled sense cRNA probe for mouse aminopeptidase A. As in Figure 3, the section was 10 m thick and was taken from a male BALB/c mouse. The section was pretreated with chloroform prior to the hybridization. No signal could be seen, indicating the specificity of the antisense hybridization signal seen in Figure 3. (magnification, 600x).
156
CONTENTS
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References
Alkemade, J. A. C.; Molhuizen, H. O. F .; Ponec, M.; Kempenaar, J. A.; Zeeuwen, P . L. J. M.; de Jongh, G . J.; van Vlijmen-W illems, I. M. J. J.; van Erp, P . E. J.; van de Kerkhof, P . C. M.; Schalkwijk, J. (1994) SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes. J. Cell Sci. 107, 23352342. Assmann, K. J. M.; van Son, J. P . H. F .; Dijkman, H. B. P . M.; Koene, R. A. P . (1992) A nephritogenic rat monoclonal antibody to mouse aminopeptidase A. Induction of massive albuminuria after a single intravenous injection. J. Exp. Med. 175, 623636. Dijkman, H. B. P . M.; Mentzel, S.; de Jong, A. S.; Assmann; K. J. M. (1995a) RNA In Situ hybridization using digoxigenin-labeled cRNA probes. Biochemica (U.S. version) No. 2 [1995], 2327.
Dijkman, H. B. P . M.; Mentzel, S.; de Jong, A. S.; Assmann; K. J. M. (1995b) RNA In Situ hybridization using digoxigenin-labeled cRNA probes. Biochemica (worldwide version) No. 2 [1995], 2125. Barton, A. J. L.; Pearson, R. C. A.; Najlerahim, A.; Harrison, P . J. (1993) Pre- and postmortem influences on brain RNA. J. Neurochem. 61, 111. Hannon, K.; Johnstone, E.; Craft, L. S.; Little, S. P .; Smith, C. K.; Heiman, M. L.; Santerre, R. F . (1993) Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization. Anal. Biochem. 212, 421427. Wu, Q.; Lahti, J. M.; Air, G . M.; Burrows, P . D.; Cooper, M. D. (1990) Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family. Proc. Natl. Acad. Sci. USA 87, 993997.

Reagent DIG RNA Labeling Kit* , **, *** EDTA Description For RNA labeling with digoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerases. Powder Cat. No. 1175 025 Pack size 1 Kit (2x10 labeling reactions) 250 g 500 g 1 kg 1g 50 g 150 U (200 l) 100 g 500 g 1 kg 3 ml (300 mg) (dilute prior to use) 3 ml (150 mg)
808 261 808 270 808 288 223 646 1 096 176 1 093 274 127 434 708 968 708 976 1 383 213
DNA, from herring sperm Blocking Reagent Anti-Digoxigenin-AP Tris
lyophilized sodium salt For nucleic acid hybridization Anti-Digoxigenin, Fab fragments conjugated with alkaline phosphatase For preparation of buffer solutions
NBT solution
100 mg/ml nitroblue tetrazolium salt in 70% (v/v) dimethylformamide 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), toluidinium salt in 100% dimethylformamide
BCIP solution
1 383 221
CONTENTS
INDEX
157
Procedures for I n Situ H ybridiz ation to C hrom osom es, C ells, and Tissue Sections W hole m ount I SH
Localization of the expression of the segmentation gene hunchback in Drosophila embryos with DIG -labeled DNA probes
D r. D . Tautz , I nstitute for G enetics and M icrobiology, U niv ersity of M unich, G erm any. I n situ hybridization to RN A on sections of early embryos provided one of the most significant advances for the analysis of spatially regulated transcripts of segmentation genes in D rosophila. It was therefore soon adapted to other systems. The procedure is, however, rather tedious and time-consuming. It depends on the quality of the sections and particularly on the quality of the probe. In contrast, antibody staining of the protein products from these genes in whole embryos simplified the procedure and allowed a much higher resolution. H owever, certain regulatory events have to be analyzed at the RN A level. Also antibody production itself is somewhat time consuming. We have therefore sought to develop an in situ hybridization technique which offers the same advantages as antibody staining. We found the use of digoxigenin-labeled probes to be highly successful for this purpose. These probes allow a resolution which is directly comparable to antibody staining in embryos, and both the sensitivity and the speed is superior to conventional radioactive methods. The method is not limited to applications in Drosophila, but is apparently suitable for all kinds of tissues, at least for those in which antibody staining has been successful. We have already developed a protocol for the staining of specific cells in whole H ydra animals, which required only minor modifications (E. Kurz and C. David, University of Munich, Dept. of Zoology). The procedure given below has been published (Tautz and Pfeiffle, 1989).
I. Preparation of embryos
N ote: A ll em bryos in this study cam e from w ild-type D rosophila flies and w ere collected on apple juice agar plates at interv als of 04 h at 25 C (W ieschaus and N sslein-Volhard, 1986)
1 Collect the embryos in small baskets. 2 Wash embryos with water. 3 Dechorionate embryos for about 23 min
in a solution of 50% commercial bleach (about 5% sodium hypochlorite) in water. 4 Wash the embryos with 0.1% Triton X 100. 5 Fix the embryos either with formald ehyde (Procedure IIA) or paraformaldehyde (Procedure IIB).
IIA. Formaldehyde fixation
1 Transfer each embryo into a glass
scintillation vial containing 4 ml of fixation buffer [0.1 M H epes, pH 6.9; 2 mM magnesium sulfate, 1 mM EGTA (from a 0.5 M stock EGTA solution that has been adjusted to pH 8 with N aO H )]. 2 Add to the scintillation vial 0.5 ml 37% formaldehyde solution and 5 ml heptane. 3 Shake the vial vigorously for 1520 min to maintain an effective emulsion of the organic and the water phase. 4 Remove the lower phase and add 10 ml methanol. If the embryos sink to the bottom at this stage, proceed to Step 5. If the embryos do not sink to the bottom at this stage, remove the upper phase (heptane) and add more methanol. 5 Store the fixed embryos in methanol (up to several weeks) in the refrigerator.
158
CONTENTS
INDEX
IIB. Paraformaldehyde fixation
III. Pretreatment
C aution: I n the steps below, use the norm al precautions for av oiding potential R N ase contam ination.
1 Treat the PBS for the following steps with
Note: This protocol is more laborious, but leads, in some cases, to a lower background and to better preservation of the morphology.
1 Transfer
the embryos into a glass scintillation vial containing 1.6 ml of fixation buffer [100 mM H epes, pH 6.9; 2 mM magnesium sulfate, 1 mM EGTA (from a 500 mM stock EGTA solution that has been adjusted to pH 8 with N aO H )]. 2 Liquify a 20% stock solution of para formaldehyde in water as follows: Remove the stock 20% paraformaldehyde from its storage at 20C. H eat the stock to 65C. N eutralize with a little N aO H . 3 From the liquified stock 20% solution of paraformaldehyde, remove 0.4 ml and add it to the scintillation vial. 4 Also add 8 ml heptane to the vial. 5 Shake the vial and treat with methanol as in Steps 3 and 4 in Procedure IIA above. 6 Transfer each embryo to a 1.5 ml micro centrifuge tube containing ME [90% methanol, 10% 0.5 M EGTA] . 7 Prepare PP solution (4% paraformaldehyde in PBS). N ote: PBS contains 130 m M N aC l and 10 m M sodium phosphate; pH 7.2.
8 Refix the embryos and dehydrate by
diethylpyrocarbonate (20 l per 500 ml PBS), then autoclave the treated PBS. 2 Place each fixed embryo in a 1.5 ml microcentrifuge tube. Perform the incubations in Steps 38 below at room temperature on a revolving wheel. 3 Wash each embryo 3 x 5 min in 1 ml PBT (PBS plus 0.1% Tween 20). Note: Tween reduces non-specific adhesion of the embryos to the plastic surfaces.
4 Incubate the embryo for 35 min in 1 ml of 5 Stop the digestion by removing the
PBS containing 50 g/ml Proteinase K.
Proteinase K solution, adding 1 ml PBT containing 2 mg/ml glycine to the embryo. Incubate for 2 min. C aution: T he proteinase digestion tim e is critical. I f it is too short, the back ground increases and sensitiv ity is lost. I f it is too long, the em bryos burst during the subsequent steps.
6 After the Proteinase K digestion, wash
7 Refix for 20 min in 1 ml PP (4%

8 Wash 3 x 10 min in 1 ml PBT.
the embryo 2 x 5 min in 1 ml PBT. paraformaldehyde in PBS).
passage through the following series of solutions containing ME and PP: 5 min in ME:PP (7:3). 5 min in ME:PP (1:1). 5 min in ME:PP (3:7). 20 min in PP alone. 9 Wash the embryos in PBS for 10 min and do either of the following: Use the fixed embryos in Procedure III. or If the fixed embryos are not to be used immediately, dehydrate them in a series of ethanol solutions (30% , 50% , then 70% ethanol) and store them at 20C. C aution: D ehydrated, stored em bryos m ust be rehydrated before they can be used in Procedure I I I .
IV . Probe labeling
The probes are DN A restriction fragments isolated from agarose gels. Label them according to the random priming method described in Chapter 4 of this manual.
CONTENTS
INDEX
159
V . Hybridization
1 Prepare the hybridization solution (H S)
VI. Detection
1 Prepare a fresh dilution (1:2000 to
[50% formamide; 5 x SSC (where 1 x SSC contains 150 mM N aCl, 15 mM sodium citrate); 50 g/ml heparin, 0.1% Tween 20; and 100 g/ml sonicated and denatured salmon sperm DN A]. N ote: H S m ay be stored at 20 C .
2 Wash the embryos as follows: 20 min in 1:1 H S:PBT. 2060 min in H S. 3 Prehybridize for 2060 min in H S at 4 H eat denature the probe in the presence
1:5000) of alkaline phosphataseconjugated anti-DIG-antibody in PBT. N ote: T he antibody m ay be preabsorbed for 1 h w ith fixed em bryos to reduce the back ground.
2 Incubate each embryo with 500 l freshly
45C in a water bath.
of 10 g sonicated salmon sperm DN A. N ote: T he salm on sperm D N A should effectiv ely com pete out sim ple sequences and thus reduce the back ground.
5 Dilute the probe to approximately 0.5 g 6 Remove most of the prehybridization
diluted antibody conjugate for 1 h at room temperature on a revolving wheel. 3 Wash the embryo as follows: 4x20 min in PBT. 3x5 min in color buffer [100 mM N aCl, 50 mM MgCl2, 100 mM Tris (pH 9.5), 1 mM levamisole, 0.1% Tween 20]. N ote: L ev am isole is a potent inhibitor of lysosom al phosphatases.
4 Bring the embryos into a small dish with
labeled probe per ml in H S.
supernatant from the embryo, then add the heat-denatured probe and thoroughly mix. 7 H ybridize overnight at 45C in a water bath without shaking. 8 Wash the embryos at room temperature in each of the following solutions: 20 min in H S. 20 min in 4:1 H S:PBT. 20 min in 3:2 H S:PBT. 20 min in 2:3 H S:PBT. 20 min in 1:4 H S:PBT. 2 x 20 min in PBT. N ote: U se a less extensiv e w ashing protocol if probe produces a low back ground.
1 ml of color buffer. Add to this 4.5 l N BT and 3.5 l BCIP with thorough mixing. 4 Develop the color for 1060 min in the dark. N ote: C olor dev elopm ent m ay occasionally be controlled under the binocular m icroscope and stopped in PBT before back ground dev elops.
6 Dehydrate the embryos in a series of
ethanol solutions and mount in GMM (Lawrence et al., 1986).
Results
Figure 1 shows typical results obtained with the above protocol. Panel a shows three embryos at different developmental stages which represent the dynamic expression pattern of hunchback. The embryo on the top right in Panel a shows the maternal gradient of hunchback RN A distribution; the embryo at the left the primary zygotic expression in two domains; and the middle embryo, the late zygotic expression. In Panel b, an enlarged section of an embryo shows the predominant cytoplasmic location of the hybridization signal. The section of the embryo corresponds to the frame depicted in Panel a although the photo was taken of a different embryo. Panel c shows an embryo after gastrulation, where individual cells of the developing nervous system express hunchback.
160
CONTENTS
INDEX
nuclei cytoplasm
yolk
References
Lawrence, P . A.; Johnston, P .; Morata, G . (1986) Methods of marking cells. In: Roberts, D. B. (ed.) Drosophila, A Practical Approach. Oxford: IRL Press, pp. 229242. Tautz, D.; Pfeiffle, C. (1989) A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback. Chromosoma (Berl.) 98, 8185. Reagent Triton Hepes X-100 Description Vicous, liquid Purity: 98% (from N) Wieschaus, E.; Nsslein-Vollhard, C. (1986) Looking at embryos. In: Roberts, D. B. (ed.) Drosophila, A Practical Approach. Oxford: IRL Press, pp. 199228.
Figure 1: In situ hybridization on whole Drosophila embryos using a probe for the segmentation gene hunchback. Optical sections were obtained by appropriate focusing. For details of the panels, see text. Panel a (The bar at the top represents approx. 100 m.) Panel b (The bar at the top represents approx. 5 m.)

Cat. No. 789 704 223 778 737 151 242 608 1 093 053 1 332 465 Pack size 100 ml 100 g 500 g 1 kg 50 g 5x10 ml 25 mg 100 mg 500 mg 1g 160 l (40 labeling reactions) 150 U (200 l) 3 ml (300 mg) (dilute prior to use) 3 ml (150 mg)
EGTA Tween 20
Purity: > 98%
Proteinase K
Lyophilizate
161 519 745 723 1 000 144 1 092 766 1 585 606
DIG-High Prime* , **, *** Premixed solution for 40 random-primed DNA labeling reactions with DIG -11-dUTP , alkali-labile Anti-Digoxigenin-AP 750 units/ml Anti-Digoxigenin, Fab fragments conjugated to alkaline phosphatase 100 mg/ml nitroblue tetrazolium salt in 70% (v/v) dimethylformamide 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), toluidinium salt in 100% dimethylformamide
1 093 274
***EP Patent 0 324 474 granted to and EP Patent application 0 371 262 pending for Boehringer Mannheim G mbH. ***This product or the use of this product may be covered by one or more patents of Boehringer Mannheim G mbH, including the following: EP patent 0 649 909 (application pending). ***Licensed by Institute Pasteur.
NBT solution
1 383 213
BCIP solution
1 383 221
CONTENTS
INDEX
161
Detection of even-skipped transcripts in Drosophila embryos with PCR/DIG -labeled DNA probes
D r. N . Patel and D r. C . G oodm an, C arnegie I nstitute of Washington, Em bryology D epartm ent, Baltim ore, M aryland, U SA .
This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. The in situ hybridization and detection was performed essentially according to the protocol of Tautz and Pfeiffle (1989). This laboratory uses PCR rather than random primed labeling to prepare DIGlabeled probes because: A much larger quantity of probe can be made with the same amount of starting nucleotide. The ratio of labeled DN A to unlabeled starting material is much higher (especially important when transcripts to be detected are not very abundant). PCR can produce strand-specific copies. Disadvantage:
It is more difficult to control the probe
I. Probe labeling
1 Prepare the following stock solutions: 10 x concentrated reaction mix:500 mM
KCl; 100 mM Tris-H Cl, pH 8.3; 15 mM MgCl2; 0.01% (w/v) gelatin. 5 x concentrated dN TP mix: 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM digoxigenin11-dUTP. A primer stock solution containing either 30 ng/l (approx. 5.3 mmol) primer 1 (e.g. generated by SP6 RN A polymerase) or 30 ng/l (approx. 5.3 mmol) primer 2 (e.g. generated by T7 RN A polymerase). 2 With a restriction enzyme, linearize dual promotor vector DN A containing the insert, as one would to make run-off RN A transcripts. N ote: T he tw o different prim ers can be used to create an antisense strand and a sense strand (as control).
3 H eat inactivates the restriction enzyme. 4 Dilute the linearized DN A with water to
size. This laboratory has also found that biotin16-dUTP incorporated in the same way and detected with streptavidin-alkaline phosphatase is about three- to fivefold less sensitive than DIG-labeled probes in in situ hybridization experiments.
a final concentration of about 100 200 ng/l. N ote: I f the insert is m uch ov er 3 k b, the probe produced w ill probably not represent the entire insert.
5 Set up the following reaction mix: 9.25 l water. 2.5 l 10 x concentrated reaction
5.0 l 5 x concentrated dN TP mix. 5.0 l primer 1 or 2 (from 30 ng/l

6 Add 40 l mineral oil, centrifuge, then
mixture.
2.0 l linearized DN A (100200 ng/l).
stock).
7 To the mix, add 1.25 l of 1 unit/l Taq
boil the mix for 5 min.
DN A Polymerase (1.25 units of Taq Polymerase). [Final volume of reaction mix with Taq is 25 ml.] 8 Mix the contents of the reaction tube and then centrifuge for 2 min.
162
CONTENTS
INDEX
9 Incubate for 30 cycles in the PCR thermal
cycler under the following conditions: Denaturation at 95C for 45 s. Annealing at 5055C for 30 s. N ote: A nnealing tem perature depends on length of prim er. U se 55C for a 21-m er. Elongation at 72C for 1.01.5 min. N ote: Elongation tim e depends on length of insert. U se 1 m in for 1.0 k b or less, 1.5 m in for 2.5 k b or m ore.
10 After the PCR run, add 75 l distilled H 2O 11 Remove 9095 l of the reaction mix
II. Evaluation of labeling reaction

1 Prepare an aliquot of the probe as follows: Remove 1 l of probe from the Add 5 l of 5 x SSC. Boil 5 min. Q uick cool on ice. Centrifuge.
reaction mix.
2 Spot 12 l of the probe aliquot onto a
to the reaction tube, then centrifuge it.
12 To recover the DN A, do an ethanol
from beneath the oil.
precipitation as follows: Add N aCl to a final concentration of 0.1 M. Add 10 g of glycogen or tRN A as a carrier (0.5 l of a 20 mg/ml stock). Add 3 volumes of 100% EtO H . Mix well and leave at 70C for 30 min. Centrifuge. Wash pellet with 70% ethanol. Dry under vacuum. O ptional: Perform a second ethanol precipitation as abov e.
13 Resuspend DN A pellet in 300 l of
small nitrocellulose strip cut to fit into a 1.5 ml microcentrifuge tube or a 5 ml snap cap tube. 3 Bake the filter between two sheets of filter paper in an 80C vacuum oven for 30 min. C aution: T he residual form am ide m ay cause the nitrocellulose to w arp. I f this is a problem , reduce the tim e in the bak ing ov en or do this spot test before the second precipitation (Procedure I , Step 12). N ote: U nincorporated nucleotide binds only slightly to the nitrocellulose.
4 Treat the baked filter as follows: Wet the filter with 2 x SSC. Wash filter 2 x 5 min in PBT (1 x PBS,
Place filter into a 1.5 ml micro5 Detect the labeled probe as follows: Block the filter by incubating it
0.2% BSA, 0.1% Triton X-100).
centrifuge tube or 5 ml snap cap tube.
hybridization buffer [50% formamide; 5 x SSC (where 1 x SSC contains 150 mM N aCl, 15 mM sodium citrate); 50 g/ml heparin, 0.1% Tween 20; and 100 g/ ml sonicated and denatured salmon sperm DN A] (according to Tautz and Pfeifle, 1989). 14 To reduce the size of the single-stranded DN A, boil the probe for 4060 min. N ote: For efficient penetration of and hybridiz ation to the em bryos, the av erage probe length should be about 50200 bp.
15 Dilute the probe as much as tenfold
Incubate in PBT for 3060 min with a
30 min in PBT.
1:2000 dilution (in PBT) of alkaline phosphatase-conjugated anti-DIG antibody. Wash 4 x 15 min in PBT. Wash 2 x 5 min in a solution containing 100 mM N aCl; 50 mM MgCl2; 100 mM Tris, pH 9.5; 0.1% Tween 20. N ote: L ev am isole is not needed.
6 Develop color with N BT and BCIP as
before use. N ote: O ptim al dilution v aries depending on the abundance of the transcript and back ground staining. We recom m end using the probe either undiluted (original 300 l) or diluted up to threefold for the initial experim ent.
described in the appropriate Boehringer Mannheim pack insert. 7 Stop the reaction when the spots are visible. N ote: Spots should be v isible w ithin a few m inutes and dark after 1015 m in.
CONTENTS
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III. Preparation of embryos, hybridization and detection

Perform the preparation of the embryos, subsequent hybridization of the DIG-PCR probe, and immunological detection as described in the Tautz article, Localization of the expression of the segmentation gene hunchback in Drosophila embryos with digoxigenin-labeled DN A probes, page 158 in this manual.
Results
a b
Reference
Tautz, D.; Pfeiffle, C. (1989) A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback. Chromosoma (Berl.) 98, 8185.
Figure 1: Detection of the even-skipped phenotype on blastoderm stage Drosophila embryos. Panel a shows an in situ hybridization using the Drosophila even-skipped gene as probe. Panel b demonstrates the even-skipped product using the specific antibody as probe. The seven stripe pattern is associated with the even-skipped phenotype.

Reagent PCR DIG Probe Synthesis Kit* Description For generating highly sensitive probes labeled with DIG -dUTP (alkali-labile) in the polymerase chain reaction (PCR) using a ratio of 1+2 (DIG -dUTP:dTTP) Cat. No. 1 636 090 Pack size 1 Kit (25 reactions)
Tween 20 Glycogen BSA Triton X-100 *This product is sold under licensing arrangements with Roche Molecular Systems and The PerkinElmer Corporation. Purchase of this product is accompanied by a license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with an Authorized Thermal Cycler. For complete license disclaimer, see inside back Anti-Digoxigenin-AP From rabbit liver Highest quality, lyophilizate Vicous, liquid 750 units/ml Anti-Digoxigenin, Fab fragments conjugated to alkaline phosphatase 100 mg/ml nitroblue tetrazolium salt in 70% (v/v) dimethylformamide 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), toluidinium salt in 100% dimethylformamide
1 332 465 106 089 238 031 238 040 789 704 1 093 274
5x10 ml 500 mg 1g 10 g 100 ml 150 U (200 l) 3 ml (300 mg) (dilute prior to use) 3 ml (150 mg)
NBT solution
1 383 213
BCIP solution
1 383 221
164
CONTENTS
INDEX
Wholemount fluorescence insitu hybridization(FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana
1
Serge Bauw ens1 and Patrick Van O ostv eldt 2 L aboratory for G enetics, G ent U niv ersity, G ent, Belgium 2 L aboratory for Biochem istry and M olecular C ytology, G ent U niv ersity, G ent, Belgium
3 Pipette onto germination medium (1x
H ybridizing fluorescently labeled DN A probes in situ to the chromatin of interphase nuclei in whole mounts allows the study of nuclear architecture in morphologically well preserved specimens. Fluorescent labeling of the DN A probes (either directly or indirectly) allows the simultaneous, but differential, detection of several sequences (e.g. Lengauer et al., 1993; N ederlof et al., 1990). Also, fluorescent signals can be imaged by confocal microscopy so that stacks of optical section images can be recorded through the whole mounts. Multiple labeling FISH on whole mounts therefore allows the study of the relative positions of different chromosomes or chromosome segments in individual interphase nuclei as well as between nuclei of related cells. In the experiments presented here, two tandemly repeated sequences, rDN A and a 500 bp repeat sequence, were hybridized to interphase nuclei of seedlings and flowers (inflorescences) of the small cruciferous plant Arabidopsis thaliana. The procedure used in the experiments, based on the protocols published by Ludevid et al. (1992) and Tautz and Pfeifle (1989), was described earlier by Bauwens et al. (1994).
Murashige and Skoog salt mixture (Flow Laboratories, USA); 0.5 g/L 2-(N morpholino)ethane sulphonic acid (MES), pH 5.7 (adjusted with 1 M KO H ); 0.8% (w/v) Bacto-agar (Difco Laboratories, USA). 4 Allow the seeds to germinate at room conditions for 4 days. 5 Transfer part of the seedlings to soil. 6 Grow plants under continuous light conditions at desk temperature. 7 H arvest flowers and inflorescences after 34 weeks.
II. Tissue fixation

1 Place approximately 3040 seedlings or a
few flowers or inflorescences in a glass vial containing: 4.365 ml fixation buffer [1.1 x PBS; 0.067 M EGTA, pH 7.5 (adjusted with N aO H )]. N ote: 10 x PBS contains 1.3 M N aC l, 0.0027 M KC l, 0.07 M N a2H PO 4, 0.03 M N aH 2PO 4; pH 7.2. 0.135 ml 37% formaldehyde (Sigma, USA). 0.5 ml DMSO . N ote: Final concentrations in v ial are 1% form aldehyde and 10% D M SO .
2 Rock the glass vial for 25 min at room
I. Seed sterilization and germination

N ote: Procedure I is based on the protocol of Valv ek ens et al. (1988).
1 Surface sterilize seeds of Arabidopsis
thaliana (C24) by immersing them as follows: 2 min in 70% (v/v) ethanol. 15 min in a solution of 5% (v/v) N aO Cl and 0.05% (v/v) Tween 20. 2 Wash seeds 5 times in sterile, distilled water.
3 Remove the fixative and rinse as follows: 2 x with 5 ml methanol. 4 x with 5 ml ethanol. 4 Discard the last ethanol wash, then cover
5 Leave sample at 20C for 24 days.
temperature.
sample with a final 5 ml ethanol.
C aution: Keeping the m aterial for longer periods of tim e in ethanol m ak es it brittle.
CONTENTS
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165
III. Labeling of the probe DNA

1 Use the following as hybridization
IV . Pretreatment
1 Remove ethanol from seedlings or flow-
probes: A mixture of three ribosomal DN A (rDN A) inserts, each cloned into pBS (I)KS+ (Stratagene, USA) from A. thaliana. The inserts contain the 5.8S, 18S and 25S rRN A genes, as well as the intergenic region (IGR) (Unfried and Gruendler, 1990; Unfried et al., 1989). A 500 bp repeat DN A sequence, cloned into pGem-2 (Promega, USA). The repeat is one of three classes of highly repetitive, tandemly arranged DN A sequences in A. thaliana (Simoens et al., 1988). 2 Label undigested samples of both probes according to the nick translation procedures in Chapter IV of this manual. Use the following labels: Label the rDN A with either DIGdUTP or fluorescein-dUTP. N ote: T he concentration of substituted nucleotide in the nick translation labeling m ixture should be the sam e for either fluorescein-dU T P or D I G -dU T P.
Label the 500 bp repeat DN A with 3 After nick translation, treat each labeled
ers (or inflorescences) and transfer material to microcentrifuge tubes. 2 Fix each sample as follows: Rinse 2 x with 1 ml ethanol. Replace ethanol with 1 ml ethanol/ xylene (1:1) and incubate for 30 min. Rinse 2 x with 1 ml ethanol. Rinse 2 x with 1 ml methanol. Replace methanol with 1 ml of a 1:1 mixture of methanol and [PBT containing 1% (v/v) formaldehyde]. Rock for 5 min. N ote: PBT contains 1 x PBS and 0.1% (v / v ) Tw een 20. 3 Post-fix sample for 25 min in 1 ml PBT containing 1% formaldehyde. 4 Remove fixative and rinse sample with 5 x1 ml PBT. 5 Wash sample 3 x 1 ml of 2 x SSC (each wash, 5 min). N ote: 1 x SSC contains 150 m M N aC l and 15 m M sodium citrate, pH 7.0.
6 Digest with RN ase A (100 g/ml in
7 Wash 3 x 5 min with 1 ml PBT. 8 Digest with Proteinase K (40 g/ml in 9 After the Proteinase K digestion, do the
2 x SSC) for 1 h at 37C.
DIG-dUTP.
PBT) for 8 min at 37C.
probe as follows: Co-precipitate 1 g of labeled DN A with 55 g of sonicated salmon sperm DN A (Sigma, USA). Redissolve probe in 25 l H 2O to a concentration of 40 ng labeled DN A per l.
following: Rinse 2 x with 1 ml PBT. Wash 2 x 2 min with 1 ml PBT. Rinse 2 x with 1 ml PBT. 10 Postfix a second time for 25 min with 1 ml PBT containing 1% formaldehyde. 11 Remove fixative and rinse 5 x with 1 ml PBT.
166
CONTENTS
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V . In situ hybridization
1 Wash each sample 10 min with 1 ml of a
VI. Pre-absorption of antibodies (for indirect detection only)

1 Prepare powdered A. thaliana root or
1:1 mixture of PBT and hybridization solution [hybridization solution contains 50% formamide (Ultra Pure from USB, USA) in 2 x SSC]. 2 Rinse sample with 2 x1 ml hybridization solution. 3 Remove the hybridization solution and add the following to each sample (to produce a final volume of 500 l of hybridization solution): 250 l formamide. 50 l 20 x SSC. Enough H 2O to make a total volume, including probes, of 500 l. 25 l of each labeled rDN A probe (for single or double labeling experiments). 25 l labeled 500 bp repeat probe (for double labeling experiments only). N ote: Each labeled probe has a final concentration of 2 ng/ l. N ote: T his incubation m ixture can be used for either a single label hybridiz ation (fluorescein-labeled probe) w ith direct detection; a single label hybridiz ation (D I G -labeled probe) w ith indirect detection; or a double label hybridiz ation (both fluorescein- and digoxigeninlabeled probes). See Procedure VIII below for details on these different types of experiments.
4 Treat the sample as follows: Denature target and probe in hybridi-
seedling extract as follows: Grind the root or seedling under liquid nitrogen. Extract the ground powder with acetone under liquid nitrogen. Decant the acetone supernatant. Let the residual acetone evaporate from the precipitate. Use the dry, powdered precipitate in the pre-absorption procedure below. 2 Dilute each detection antibody, in 4 x SSC containing 1% (w/v) BSA, to the working dilution suggested by the manufacturers and a final volume of 500 l. 3 Add approximately 2 mg of powdered A. thaliana root or seedling extract (from Step 1 above) to the diluted antibody. 4 Pre-absorb the antibodies overnight at 15C, in the dark. 5 Centrifuge the pre-absorption mixture. 6 Use the supernatant in the immunocyto chemical detection reaction.
VII. Posthybridization washes

1 After overnight hybridization at 37C
Place immediately on ice for 3 min. Centrifuge very briefly. Incubate overnight at 37C to hybridize
zation solution for 4 min at 100C.
probe and target. C aution: I f using a directly labeled (i.e., fluorescent) probe, perform the hybridiz ation incubation and the rest of the procedure in the dark .
(Procedure V, Step 4), treat the hybridization sample as follows: Remove the hybridization solution. Wash the sample for 1 h in fresh hybridization solution at 37C. Wash the sample 4 x 30 min in hybridization solution at 37C. If performing an in situ hybridization experiment with directly labeled probe DN A (rDN A) in a single labeling experiment, proceed to procedure VIII a. If performing an in situ hybridization experiment requiring indirect detection with antibodies, proceed to step 2. 2 Bring the seedlings or flowers (or inflo rescences) gradually to 4 x SSC through the following washes (all at room temperature): 20 min with a 3:1 mix of hybridization solution and 4 x SSC. 20 min with a 1:1 mix of hybridization solution and 4 x SSC. 20 min with a 1:3 mix of hybridization solution and 4 x SSC. 4 x 5 min with 4 x SSC.
CONTENTS
INDEX
167
VIII. Immunocytochemical detection

Use the immunocytochemical detection schemes detailed in Table 1 to analyze the single and double labeling in situ hybridization experiments. Antibody a 1st (detecting) 2nd (amplifying) Fluorescein-conjugated anti-DIG antibody (from sheep)b Tetramethylrhodamine-conjugated anti-DIG antibody (from sheep)d Fluorescein-conjugated anti-sheep antibody (from donkey)c Tetramethylrhodamine-conjugated anti-sheep antibody body (from rabbit)e
VIIIb. Indirect detection
1 After the posthybridization washes (Pro-
Type of Experiment Single label, direct detection Single label, indirect detection Double label
Probe rDN A rDN A
Label FluoresceindUTP DIG-dUTP
Procedure to Follow VIIIa VIIIb
rDN A 500 bp
FluoresceindUTP DIG-dUTP
VIIIb
Table 1: Immunocytochemical detection schemes for single and double labeling experiments a All antibodies should be preabsorbed, according to Procedure VI above. b Fab fragments. c Fab fragments, from Sigma, USA. d Fab fragments. e Whole molecule, from Chemicon, USA.
VIIIa. Direct detection

1 Bring the samples to 1 x PBS gradually
through the following washes (all at room temperature): 20 min with a 3:1 mix of hybridization solution and 1 x PBS. 20 min with a 1:1 mix of hybridization solution and 1 x PBS. 20 min with a 1:3 mix of hybridization solution and 1 x PBS. 4 x 15 min in 1 ml 1 x PBS. 2 Proceed to the staining and mounting procedure (Procedure IX).
cedure VII), remove the 4 x SSC solution from the samples. 2 Add the first pre-absorbed antibody (from Table 1) to the sample and incubate overnight at 15C in the dark. 3 Remove the first antibody and wash the material 4 x15 min in 1 ml 4 x SSC containing 0.05% (v/v) Tween 20. 4 Depending on whether you are using an amplifying (second antibody), do either of the following: If you use an amplifying antibody, proceed to Step 5. If you do not use an amplifying antibody, proceed to Step 7. 5 If amplifying the signal, wash the sample 4 x 15 min with 4 x SSC containing 1% bovine serum albumin. 6 Remove the wash solution; add the second pre-absorbed antibody (from Table 1) to the sample, and incubate overnight at 15C in the dark. 7 After the last (first or second) antibody incubation, remove the antibody and wash the sample 4 x 15 min in 1 ml 1 x PBS. 8 Proceed to the staining and mounting procedure (Procedure IX).
168
CONTENTS
INDEX
IX. Staining and mounting

1 Counterstain the sample as follows: For single labeling experiments with
Xb. Confocal fluorescence microscopy

1 Record
fluorescein-conjugated antibodies or nucleotides: Counterstain the chromatin of the interphase nuclei with 0.5 g/ml propidium iodide (in 1 x PBS) for 1 h in the dark. For double labeling experiments with fluorescein- and tetramethylrhodamineconjugated nucleotides and antibodies: Counterstain the chromatin of the interphase nuclei with 0.2 g/ml DAPI (in 1 x PBS) for 1 h in the dark. N ote: D A PI is 4,6'-diam idino-2phenylindole. 2 Place a few seedlings or flowers (or part of an inflorescence) on a slide. 3 Apply some tape to the slides to create a support for the cover slip, so as not to crush the seedlings or flowers. 4 Apply a drop of antifade reagent (Vectashield, Vector Laboratories, USA) and cover with a cover slip.
images with the MRC-600 Confocal Scanning Laser Microscope (CSLM) System (Bio-Rad, USA). Attach the CSLM to the DIAPH O T 300 inverted microscope fitted with appropriate lenses (same microscope and lenses as described in Procedure Xa, Step 1 above). 2 Use the K1/K2 filter block combinations (Bio-Rad, USA) and either: The 568 nm line from a KryptonArgon laser (Ion Laser Technology, Utah, USA), to image the propidium iodide-stained interphase nuclei and the tetramethylrhodamine-labeled hybrids. The 488 nm line from the same Krypton-Argon laser, to image the fluorescein-labeled hybrids.

Figures 1 and 2 show some of the results obtained by FISH of rDN A and the 500 bp repeat on whole mounts of flowers and seedlings from A. thaliana. The rDN A from A. thaliana covers about 5.7 Mbp per haploid genome (Meyerowitz and Pruitt, 1985), and is distributed over two large tandem repeats on chromosomes 2 and 4 (Maluszynska and H eslop-H arrison, 1991; Murata et al. 1990). Diploid interphase nuclei from A. thaliana exhibit, however, a number of rDN A-loci ranging from two to more than four (Bauwens et al. 1991) as can clearly be seen from the merged image in Figure 1. The 500 bp repeat sequence covers about 0.30.6 Mbp per haploid genome (Bauwens et al., 1991; Simoens et al., 1988) and exhibits a chromosome-specific large cluster (Bauwens and Van O ostveldt, 1991; Bauwens et al., 1991), resulting in two distinct signals in diploid interphase nuclei as can be seen from the merged image in Figure 2. Some helpful remarks should be made about confocal observation of fluorescent signals in whole mounts.
X. Fluorescence microscopy
Xa. Conventional fluorescence microscopy
1 Visually
inspect the slides on a DIAPH O T 300 inverted microscope (N ikon, Japan), fitted with either a 60 x, N A 1.40 oil immersion lens (O lympus, Japan) or an N PL FLUO TAR, 40 x, N A 1.30 oil immersion lens (Leitz, FRG). 2 Use the following filter combinations (Chroma Technology Corp., USA): To localize propidium iodide-stained nuclei and tetramethylrhodaminelabeled hybrids: filter block 31014 404. To localize DAPI-stained nuclei: filter block 31000 404. To localize fluorescein-labeled hybrids: filter block 31 001 404. 3 As light source, use a mercury arc lamp (100 W).
CONTENTS
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169
Sequential excitation with the 568 nm and the 488 nm lines of the Kr-Ar-laser and the K1/K2 filter combinations from the BioRad MRC-600 CSLM, allowed a clear separation of the red (tetramethylrhodamine) and the green (fluorescein) signal. [See the signals from the 500 bp repeat (red) and the rDN A (green) probes in Figure 2.]. Especially, the yellow 568 nm line allows specific excitation of the red fluorescing dyes such as tetramethylrhodamine or, preferably, Texas Red with almost no excitation of fluorescein. Confocal observation of the preparations appeared to be absolutely necessary to obtain clear images of the signals, especially in the very dense meristematic tissues of the seedling roottips and the developing flowers in the inflorescences. This was especially true for the weaker signal of the 500 bp repeat that, in many cases, could not be observed through the autofluorescence haze by conventional fluorescence microscopy. Acquiring digital images through confocal microscopy has the added advantage that images recorded at different wavelengths can be easily and accurately merged and compared. Finally, the development of FISH protocols on whole mounts for localizing mRN A sequences, as already suggested by de Almeida Engler et al. (1994), will make it possible to follow the expression of different genes simultaneously at the cellular level, in three dimensions, through confocal observation.
Figure 1: Whole mount FISH with rDNA on a developing flower from A. thaliana. The image is a merged image representing part of the pistil of a developing flower, showing fluorescein-labeled rDNA loci (yellow-green) and propidium iodidestained interphase nuclei (red). An extended focus image of 15 optical sections through the pistil was created by maximum brightness projection before merging the green (rDNA) and red (nuclei) = 17 mm. images. Bar 25 m ^
5
Figure 2: Whole mount double labeling FISH with rDNA and the 500 bp repeat on a seedling from A. thaliana. This merged image represents part of the meristematic zone of a seedling roottip. It shows fluorescein-labeled rDNA loci (green) and tetramethylrhodamine-labeled 500 bp repeat loci (red). An extended focus image of 20 optical sections through the meristematic zone of the roottip was created by maximum brightness projection before merging the green (rDNA) and red (500 bp repeat) = 15 mm. images. Bar 25 m ^
References
Bauwens, S.; Katsanis, K.; Van Montagu, M.; Van Oostveldt, P .; Engler, G . (1994) Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana. Plant J. 6, 123131. Bauwens, S.; Van Oostveldt, P . (1991) Cytogenetic analysis on interphase nuclei with non-isotopic in situ hybridization and confocal microscopy. Med. Fac. Landbouww. Rijksuniv. G ent. 56/3a, 753758. Bauwens, S.; Van Oostveldt, P .; Engler, G .; Van Montagu, M. (1991) Distribution of the rDNA and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana. Chromosoma 101, 4148. de Almeida Engler, J.; Van Montagu, M.; Engler, G . (1994) Hybridization in situ of whole-mount messenger RNA in plants. Plant Mol. Biol. Reporter 12, 321331.
170
CONTENTS
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Lengauer, C.; Speicher, M. R.; Popp, S.; Jauch, A.; Taniwaki, M.; Nagaraja, R.; Riethman, H. C.; Donis-Keller, H.; DUrso, M.; Schlessinger, D.; Cremer, T . (1993) Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple Y AC clones and whole chromosome en. 2, 505512. painting probes. Hum. Mol. G Ludevid, D.; Hfte, H.; Himelblau, E.; Chrispeels, M. J. (1992) The expression pattern of the tonoplast intrinsic protein -TIP in Arabidopsis thaliana is correlated with cell enlargement. Plant Physiol. 100, 16331639. Maluszynska, J.; Heslop-Harrison, J. S. (1991) Localization of tandemly repeated DNA sequences in Arabidopsis thaliana. Plant J. 1, 159166. Meyerowitz, E. M.; Pruitt, R. E. (1985) Arabidopsis thaliana and plant molecular genetics. Science 229, 12141218. Murata, M.; Varga, F .; Maluszynska, J.; G ruendler, P .; Schweitzer, D. (1990) Chromosomal localization of the ribosomal RNA genes in Arabidopsis thaliana by in situ hybridization. In: Schweitzer, D.; Peuker, K.; Loidl, J. (Eds) Fourth International Conference on Arabidopsis Research, Vienna, Abstracts, 5.
Nederlof, P . M.; van der Ploeg, S.; Wiegant, J.; Raap, A. K.; Tanke, H. J.; Ploem, J. S.; van der Ploeg, M. (1990) Multiple fluorescence in situ hybridization. Cytometry 11, 126131. Simoens, C. R.; G ielen, J.; Van Montagu, M.; Inz, D. (1988) Characterization of highly repetitive sequences of Arabidopsis thaliana. Nucleic Acids Res. 16, 67536766. Tautz, D.; Pfeifle, C. (1989) A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 8185. Unfried, I.; G ruendler, P . (1990) Nucleotide sequence of the 5.8S and 25S rRNA genes and of the internal transcribed spacers from Arabidopsis thaliana. Nucleic Acids Res. 18, 4011. Unfried, I.; Stocker, U.; G ruendler, P . (1989) Nucleotide sequence of the 18S rRNA gene from Arabidopsis thaliana Co 10. Nucleic Acids Res 7, 7513. Valvekens, D.; Van Lijsebettens, M.; Van Montagu, M. (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 55365540.

Reagent DIG-Nick Translation Mix* Nick Translation Mix* Description Cat. No. Pack size 160 l For generation of highly sensitive probes for 1745 816 in situ hybridization with digoxigenin-11-dUTP . Premixed solution for 40 labeling reactions For generation of highly sensitive probes for 1745 808 fluorescence in situ hybridization. The Nick Translation Mix for in situ probes is designed for direct fluorophore-labeling of in situ probes. Tetralithium salt, solution, 1 mmol/l Set of dATP , dCTP , dG TP , dTTP , lithium salts, solutions Fab Fragments from sheep Fab Fragments from sheep 1 373 242 1 277 049
200 l
Fluorescein-12-dUTP dNTP Set
25 nmol (25 l) 1 set, 4x10 mol (100 l) 200 g 200 g 5x10 ml 50 g 25 mg 100 mg 500 mg 1g 25 mg 100 mg
Anti-DigoxigeninRhodamine Anti-DigoxigeninFluorescein Tween 20 EGTA Proteinase K
1 207 750 1 207 741 1 332 465
Purity: > 98% Lyophilizate
1 093 053 161 519 745 723 1 000 144 1 092 766 109 142 109 169
RNase A
Dry powder
*This product or the use of this product may be covered by one or more patents of Boehringer Mannheim G mbH, including the following: EP patent 0 649 909 (application pending).
CONTENTS
INDEX
171
References
CONTENTS
INDEX
R eferences
In this chapter general references of publications on the use of digoxigenin in in situ hybridization experiments are listed alphabetically to give to the reader an overview on the wide range of applications presently available.
1. mRNA target
A. DNA probe
a. RPL
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174
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b. Nick translation
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c. cDNA synthesis
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d. PCR
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B. RNA probes
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180
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C. O ligonucleotide probes
a. 3'-Endlabeling
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b. 3'-Tailing
Artero, R. D.; Akam, M.; Prez-Alonso, M. (1992) Oligonucleotide probes detect splicing variants in situ in Drosophila embryos. Nucl. Acids Res. 20 (21), 56875690. Amberg, D. C.; G oldstein, A. L.; Cole, C. N. (1992) Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. G enes Develop. 6, 11731189. Baldino, F .; Lewis, M. E. (1989) Non-radioactive in situ hybridization histochemistry with digoxigenindUTP labeled oligonucleotides. Methods in Neuroscience Vol. 1 (Conn, P . M., ed.) Acadmic Press, New Y ork, 282292. Black, M.; Carey, F . A.; Farquharson, M. A.; Murray, G . D.; McNicol, A. M. (1993) Expression of the proopiomelanocortin gene in lung neuroendocrine tumours: in situ hybridization and immunohistochemical studies. J. Pathol. 169, 329334. Brouwer, N.; Van Dijken, H.; Ruiters, M. H. J.; Van Willigen, J.-D.; Ter Horst, G . J. (1992) Localization of dopamine D2 receptor mRNA with non-radioactive in situ hybridization histochemistry. Neurosci. Letters 142, 223227. Dechesne, C. J.; Winsky, L.; Moniot, B.; Raymond, J. (1993) Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes. Hear. Res. 69, 9197.
CONTENTS
INDEX
183
R eferences
Dickerson, D. S.; Huerter, B. S.; Morris, S. J.; Chronwall, B. M. (1994) POMC mRNA levels in individual melanotropes and G FAP in glial-like cells in rat pituitary. Peptides 15 (2), 247256. Dirks, R. W .; Van Dorp, A. G . M.; Van Minnen, J.; Fransen, J. A. M.; Van der Ploeg, M.; Raap, A. K. (1992) Electron microscopic detection or RNA sequences by non-radioactive in situ hybridization in the mollusk lymnaea stagnalis. J. Histochem. Cytochem. 40, 16471657. Dirks, R. W .; Van G ijlswijk, R. P . M.; Vooljs, M. A.; Smit, A. B.; Bogerd, J.; Van Minnen, J.; Raap, A. K.; Van der Ploeg, M. (1991) 3End fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple, simultaneous RNA detection. Experim. Cell Res. 194, 310315. Dirks, R. W .; Van der Rijke, F . M.; Fujishita, S.; Van der Ploeg, M.; Raap, A. K. (1993) Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes. J. Cell Sci. 104, 11871197. Dumas, S.; Horellou, P .; Helin, C.; Mallet, J. (1992) Co-expression of tyrosine hydroxylase messenger RNA1 and 2 in human ventral mesencephalon revealed by digoxigenin- and biotin-labeled oligodeoxyribonucleotides. J. Chem. Neuroanatomy 5, 1118. Crabb, I. D.; Hughes, S. S.; Hicks, D. G .; Puzas, J. E.; Tsao, G . J. Y .; Rosier, R. N. (1992) Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Am. J. Pathol. 141 (3), 579589. Farquharson, M.; Harvie, R.; McNicol, A. M. (1990) Detection of messenger RNA using a digoxigenin end labeled oligonucleotide probe. J. Clin Pathol. 43, 424428. Farquharson, M.; Harvie, R.; McNicol, A. M. (1990) Detection of pro-opiomelanocortin messenger RNA using adigoxigenin end labeled oligodeoxynucleotide probe. J. Endocrinol. 124 (Supplement). Fevre-Montange, M.; Trembleau, A.; Landry, M.; Calas, A. (1992) Hybridation in situ avec des sondes oligonuclotidiques marques la digoxignine: application la dtection des ARNm de la vasopressine et de locytocine. Techniques, 479486. G rega, D.; Cavanaugh, T .; Martin, R.; Lewis, M.; Baldino, F . (1989) In situ hybridization histochemistry using a new non-radioactive detection method. ICSU Short Reports, Advances in G ene Technology: Molecular Neurobiology and Neuropharmacology, Proceedings of the 1989 Miami Bio/Technology Winter Symposium, Vol. 9, p. 69, IRL Press, McLean, Virginia. G uellec Le, P .; Dumas, S.; Volle, G . E.; Pidoux, E.; Moukhtar, M. S.; Treilhou-Lahille, F . (1993) An efficient method to detect calcitonin mRNAin normal and neoplastic rat c-cells (Medullary Thyroid Carcinoma) by in situ hybridization using a digoxigenin-labeled synthetic oligodeoxyribonucleotide probe. J. Histochem. Cytochem. 41, 389395. Hamada, K.; Okawara, Y .; Fryer, J. N.; Tomonaga, A.; Fukuda, H. (1994) Localization of mRNA of procollagen 1 type I in torn supraspinatus tendons. Clin. Orthopaed. Related Res. 304, 1821.
Harper, S. J.; Pringle, J. H.; G illies, A.; Allen, A. C.; Layward, L.; Feehally, J.; Lauder, I. (1992) Simultaneous in situ hybridization of native mRNA and immunoglobulin detection by conventional immunofluorescence in paraffin way embedded sections. J. Clin. Pathol. 45, 114119. Harvie, R.; Elvin, P .; McArdle, C.; Morten, J. E. N.; McNicol, A. M. (1991) Detection of mRNA for ribosomal phosphoprotein P2 in normal colon and colonic tumours by in situ hybridization. J. Pathol. 164, 6773. Hilton, D. A.; Day; C.; Pringle, J. H.; Fletcher, A.; Chambers, S. (1992) Demonstration of the distribution of Coxsackie virus RNAin neonatal mice by non-isotopic in situ hybridization. J. Virol. Meth. 40, 155162. Hilton, D. A.; Fletcher, A.; Pringle, J. H. (1994) Absence of coxsacki viruses in idiopathic inflammatory muscle disease by in situ hybridization. Neuropathol. Appl. Neurobiol. 20, 238242. Humpel, C.; Lindqvist, E.; Olson, L. (1993) Detection of nerve growth factor mRNA in rodent salivary glands with digoxigenin- and 33P-labeled oligonucleotides: effects of castration and sympathectomy. J. Histochem. Cytochem. 41 (5), 703708. Kendall, C. H.; Roberts, P . A.; Pringle, J. H.; Lauder, I. (1991) The expression of parathyroid hormone messenger RNA in normal and abnormal parathyroid tissue. J. Pathol. 165, 111118. Kennedy, R. E.; Hutchins, J. B. (1992) Choline acetyltransferase expression studied with an oligonucleotide probe. Cell. Mol. Neurobiol. 12 (4), 309315. Khan, G .; Coates, P .J.; Kangro, H. O.; Slavin, G . (1992) Epstein Barr virus (EBV) encoded small RNAs: Targets for detection by in situ hybridization with oligonucleotide probes. J. Clin. Pathol. 45, 616620. Khan, G .; Norton, A. J.; Slavin, G . (1993) EpsteinBarr virus in angioimmunoblastic T-cell lymphomas. Histophatol. 22, 145149. Kirchgessner, A. L.; Liu, M.-T .; Howard, M. J.; G ershon, M. D. (1993) Detection of the 5-HT1A Receptor and 5-HT1A Receptor mRNA in the rat bowel and pancreas: comparison with 5-HT1P Receptors. J. Comparative Neurol. 327, 233250. Koji, T .; Brenner, R. M. (1993) Localization of estrogen receptor messenger ribonucleic acid in rhesus monkey uterus by nonradioactive in situ hybridization with digoxigenin-labeled oligodeoxynucleotides. Endocrinol. 132, 382392. Komminoth, P . (1992) Digoxigenin as an alternative probe labeling for in situ hybridization. Diagn. Mol. Pathol. 1 (2), 142150. Komminoth, P .; Merk, F . B.; Leav, I.; W olfe, H. J.; Roth, J. (1992) Comparison of 35S- and digoxigeninlabeled RNA and oligonucleotide probes for in situ hybridization. Histochem. 98, 217228. Krizbai, I.; Deli, M.; Lengyel, I.; Maderspach, K.; Pkski, M.; Jo, F .; W olff, J. R. (1993) In situ hybridization with digoxigenin labeled oligonucleotide probes: detection of CAMK-II gene expression in primary cultures of cerebral endothelial cells. Neurobiol. 1 (3), 235240.
184
CONTENTS
INDEX
R eferences
7. Apoptosis research using DIG -dUTP and terminal transferase

Billig, H.; Furuta, I.; Hsueh, A. J. (1993) Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinol. 133 (5) 22042212. Billig, H.; Furuta, I.; Hsueh, A. J. W . (1994) G onadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: Biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinol. 134 (1), 245252. G old; R.; Schmied, M.; Rothe, G .; Zischler, H.; Breitschopf, H.; W ekerle, H.; Lassmann, H. (1993) Detection of DNA fragmentation in apoptosis: Application of in situ nick translation to cell culture systems and tissue sections. J. Histochem. Cytochem. (in press). G orczyca, W .; Bigman, K.; Mittelman, A.; Ahmed, T .; G ong, J.; Melamed, M. R.; Darzynkiewicz, Z. (1993) Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7 (5), 659670. G orczyca, W .; G ong, J.; Ardelt, B.; Traganos, F .; Darzynkiewicz, Z. (1993) The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res. 53, 31863192. G orczyca, W .; G ong, J.; Darzynkiewicz, Z. (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Canc. Res. 53, 19451951. Sgonc, R.; Boeck, G .; Dietrich, H.; G ruber, J.; Reicheis, H.; Wick, G . (1994) Simultaneous determination of cell surface antigens and apoptosis. TIG 10 (2), 4142.
8. Miscellaneous
Schfer, C.; Mller, M.; Leitner, M. D.; W achtler, F . (1993) The uptake of uridine in the nucleolus occurs in the dense fibrillar component. Cytogenet. Cell G enet. 64, 2730. W ansink, D. G .; Manders, E. E. M.; van der Kraan, I.; Aten, J. A.; van Driel, R.; de Jong, L. (1994) RNA polymerase II transcription is concentrated outside replication domains throughout S-phase. J. Cell Sci. 107, 14491456.
200
CONTENTS
INDEX
R eferences
Krusche, C.; Beier, H. M. (1994) Localization of uteroglobin mRNA by nonradioactive in situ hybridization in the pregnant rabbit endometrium. Ann. Anat. 176, 2331. Laverdure, A.-M.; Carette-Desmoucelles, C.; Breuzet, M.; Descamps, M. (1994) Neuropeptides and related nucleic acid sequences detected in peneid shrimps by immunohistochemistry and molecular hybridizations. Neurosci. 60 (2), 569579. Laverdure, A.-M.; Breuzet, M.; Soyez, D.; Becker, J. (1992) Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in homarus americanus by in situ hybridien. Compar. Endocrinol. 87, 443450. zation. G Lee, D.; Huang, W .; Y ang, Z.; Copolov, D. L.; Lim, A. T . (1992) In vitro evidence for modulation of morphological and functional development of hypothalamic immunoreactive atrial natriuretic peptide neurons by cyclic 3', 5'-adenosine monophosphate. Endocrinol. 131, 911918. Lewis, M. E.; Robbins, E.; G rega, D.; Baldino, F . (1989) Nonradioactive detection of vasopression and somatostatin mRNA with Digoxigenin-labeled oligonucleotide probes. Ann. NY Acad. Sci. V: Molecular Biology: The Future of Neuropeptide Research, pp 246253. Matute, C.; W ahle, P .; G utirrez-Igarza, K.; Albus, K. (1993) Distribution of neurons expression substance P receptor messenger RNA in immature and adult cat visual cortex. Exp. Brain Res. 97, 295300. Mertani, H. C.; W aters, M. J.; Jambou, R.; G ossard, F .; Morel, G . (1994) G rowth hormone receptor inding protein in rat anterior pituitary. Neuroendocrinol. 59, 483494. Miranda, R. C.; Toran-Allerand, C. D. (1992) Developmental expression of estrogen receptor mRNA in the rat cerebral cortex: A nonisotopic in situ hybridization histochemistry study. Cerebral Cortex Jan/Feb 2, 115. Mitchel, V .; G ambiez, A.; Beauvillain, J. C. (1993) Fine-structural localization of proenkephalin mRNAs in the hypothalamic magnocellular dorsal nucleus of the guinea pig: a comparison of radioisotopic and enzymatic in situ hybridization methods at the lightand electron-microscopic levels. Cell Tissue Res. 274, 219228. Millar, M. R.; Sharpe, R. M.; Maguire, S. M.; Saunders, P .T . K. (1993) Cellular localisation of messenger RNAs in rat testis: application of digoxigenin-labeled ribonucleotide probes to embedded tissue. Cell Tissue Res. 273, 269277. Miyazaki, M.; Nikolic-Paterson, D. J.; Masayuki, E.; Nomoto, Y .; Sakai, H.; Atkins, R. C.; Koji, T . (1994) A sensitive method of non-radioactive in situ hybridization for mRNA localization within human renal biopsy specimens: use of digoxigenin labeled oligonucleotides. Int. Med. 33, 8791. Murray, G . I.; Paterson, P . J.; Ewen, S. W . B.; Melvin, W .T . (1992) In situ hybridization of albumin mRNA in normal liver and hepatocellular carcinoma with a digoxigenin labeled oligonucleotide probe. J. Clin. Pathol. 45, 2124.
Myatt, N.; Coghill, G .; Morrison, K.; Jones, D.; Cree, I. A. (1994) Detection of tumour necrosis factor- in sarcoidosis and tuberculosis granulomas using in situ hybridization. J. Clin. Pathol. 47, 423426. Nakagawa, M.; Kukita, T .; Nakasima, A.; Kurisu, K. (1994) Expression of the type I collagen gene in rat periodontal ligament during tooth movement as revealed by in situ hybridization. Archs oral Biol. 39 (4), 289294. Negro, F .; Papotti, M.; Pacchioni, D.; G alimi, F .; Bonino, F .; Bussolati, G . (1994) Detection of human androgen receptor mRNA in hepatocellular carcinoma by in situ hybridization. Liver 14, 213219. Nicoloso, M.; Caizergues-Ferrer, M.; Michot, B.; Azum, M.-C.; Bachellerie, J.-P . (1994) U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals. Mol. Cell. Biol. 14 (9), 57665776. OKeefe, R. J.; Puzas, J. E.; Loveys, L.; Hicks, D. G .; Rosier, R. N. (1994) Analysis of type II and type X collagen synthesis in cultured growth plate chondrocytes by in situ hybridization: rapid induction of type X collagen in culture. J. Bone Mineral Res. 9 (11), 17131722. Ong, W .Y .; G arey, L. J.; Sumi, Y . (1994) Distribution of preprosomatostatin mRNA in the rat parietal and temporal cortex. Mol. Brain Res. 23, 151156. Pacchioni, D.; Papotti, M.; Andorno, E.; Bonino, F .; Mondardini, A.; Oliveri, F .; Brunetto, M.; Ussolati, G .; Negro, F . (1993) Expression of estrogen receptor mRNA in tumorous and non-tumorous liver tissue as detected by in situ hybridization. J. Surgical Oncol. Suppl. 3, 1417. Papotti, M.; Pacchioni, D.; Negro, F .; Bonino, F .; Bussolati, G . (1994) Albumin gene expression in liver tumors: diagnostic interest in fine needle aspiration biopsies. Modern Pathol. 7 (3), 271275. Paulmyer-Lacroix, O.; Anglade, G .; G rino, M. (1994) Insulin-induced hypoglycaemia increase colocalization of corticotrophin-releasing factor and arginine vasopressin mRNAs in the rat hypothalamic paraventricular nucleus. J. Mol. Endocrinol. 13, 313320. Samoszuk, M.; Nansen, L. (1990) Detection of Interleukin-5 messenger RNA in Reed-Sternberg cells of Hodgkins desease with eosinophilia. Blood 75 (1), 1316. Sharara, F . I.; Nieman, L. K. (1994) Identification and cellular localization of growth hormone receptor gene expression in the human ovary. J. Clin. Endocrinol. Metabolism 79 (2), 670672. Shorrock, K.; Roberts, P .; Pringle, J. H.; Lauder, I. (1991) Demonstration of insulin and glucagon mRNA in routinely fixed and processed pancreatic tissue by in situ hybridization.J. Pathol. 165, 105110. Thomas, G . A.; Davies, H. G .; Williams, E. D. (1994) Site of production of IG F1 in the normal and stimulated mouse thyroid. J. Pathol. 173, 355360.
CONTENTS
INDEX
185
R eferences
Throsby, M.; Lee, D.; Huang, W .; Y ang, Z.; Copolov, D. L.; Lim, A. T . (1991) Evidence for atrial natriuretic peptide-(5-28) production by macrophages of the rat spleen: An immunochemical and nonradioactive in situ hybridization approach. Endo. 129, 9911000. Throsby, M.; Y ang, Z.; Lee, D.; Huang, W .; Copolov, D. L.; Lim, A. T . (1993) In vitro evidence for atrial natriuretic factor-(5-28) production by macrophages of adult rat thymi. Endocrinol. 132 (5), 21842190. Tokushige, E.; Ito, K.; Ushikai, M.; Katahira, S.; Fukuda, K. (1994) Localization of IL-1 mRNA and cell adhesion molecules in the maxillary sinus mucosa of patients with chronic sinusitis. Laryngoscope 104, 12451250. Toran-Allerand, C. D.; Miranda, R. C.; Hochberg, R. B.; MacLusky, N. J. (1992) Cellular variations in estrogen receptor mRNA translation in the developing brain: evidence from combined [125I] estrogen autoradiography and non-isotopic in situ hybridization histochemistry. Brain Res. 576, 2541. Trembleau, A.; Roche, D.; Calas, A. (1993) Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section. J. Histochem. Cytochem. 41, 489498. Trembleau, A.; Morales, M.; Bloom, F . E. (1994) Aggregation of vasopressin mRNA in a subset of axonal swellings of the median eminence and posterior pituitary: Light and electron microscopic evidence. J. Neurosci. 14 (1), 3953. Ueyama, T .; Houtani, T .; Nakagawa, H.; Baba, K.; Ikeda, M.; Y amashita, T .; Sugimoto, T . (1994) A subpopulation of olivocerebellar projection neurons express neuropeptide Y . Brain Res. 634, 353357. W ani, G .; W ani, A. A.; Ambrosio, S. (1992) In situ hybridization of human kidney tissue reveals celltype-specific expression of the O6-methylguanineDNA methyltransferase gene. Carcinogenesis 13 (3), 463468. W ani, G .; W ani, A. A.; Ambrosio, S. (1993) Cell typespecific expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human liver tissues as revealed by in situ hybridization. Carcinogenesis 14 (4), 737741. Whitman, G .F .; Pantazis, C. G . (1991) Cellular localization of mllerian inhibiting substance messenger ribonucleic acid during human ovarian follicular ynecol. 165, development. Am. J. Obstet. G 18811886. W oodroofe, M. N.; Cuzner, M. L. (1993) Cytokine mRNA expression in inflammatory multiple sclerosis lesions: detection by non-radioactive in situ hybridization. Cytokine 5 (6), 583588. Xerri, L.; Monges, G .; G uigou, V .; Parc, P .; Hassoun, J. (1992) Detection of gastrin mRNA by in situ hybridization using radioactive- and digoxigeninlabeled probes: a comparative study. APMIS 100, 949953.
Y amada, S.; Takahashi, M.; Hara, M.; Sano, T .; Aiba, T .; Shishiba, Y .; Suzuki, T .; Asa, S. L. (1994) G rowth hormone and prolactin gene expression in human densely and sparsely granulated somatotroph adenomas by in situ hybridization with digoxigeninlabeled probes. Diagn. Mol. Pathol. 3 (1), 4652. Y oung, W . S. III (1989) Simultaneous use of digoxigenin- and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides (Edinburgh) 18, 271275. Y oung, W . S. III; Horvath, S.; Palkovits, M. (1990) The influence of hyperosmolality and synaptic inputs on galanin and vasopressin expression in the hypothalamus. Neurosci. 39 (1), 115125. Y oung, W . S. III; Reynolds, K.; Shepard, E. A.; G ainer, H.; Castel, M. (1990) Cell-specific expression of the rat oxytocin gene in transgenic mice. J. Neuroendocrinol. 2 (6), 917925. Zupanc, G . K. H.; Okawara, Y .; Zupanc, M. M.; Fryer, J. N.; Maler, L. (1991) In situ hybridization of putative somatostatin mRNA in the brain of electric gymnotiform fish. Neuro Report 2, 707710.
c. Chemical synthesis
Hassall, C. J. S.; Stanford, S. C.; Burnstock, G .; Buckley, N. J. (1993) Co-expression of four muscarinic receptor genes by the intrinsic neurons of the rat and guinea-pig heart. Neurosci. 56 (4), 10411048. Hell, K.; Pringle, J. H.; Hansmann, M.-L.; Lorenzen, J.; Colloby, P .; Lauder, I.; Fischer, R. (1993) Demonstration of light chain mRNA in hodgkins disease.J. Pathol. 171, 137143. Hodges, E.; Howell, W . M.; Tyacke, S. R.; W ong, R.; Cawley, M. I. D.; Smith, J. L. (1994) Detection of T-cell receptor chain mRNAin frozen and paraffinembedded biopsy tissue using digoxigenin-labeled oligonucleotide probes in situ. J. Pathol. 174, 151158. Li, D.; Bell, J.; Brown, A.; Berry, C. L. (1994) The observation of angiogenin and basic fibroblast growth factor gene expression in human colonic adenocarcinomas, gastric adenocarcinomas, and hepatocellular carcinomas. J. Pathol. 172, 171175. Shanks, J. H.; Lappin, T . R. J.; Hill, C. M. In situ hybridization for erythropoietin messenger RNA using digoxigenin-labeled oligonucleotides. Annals New Y ork Academy of Sciences. Thomas, G . A.; Neonakis, E.; Davies, H. G .; Wheeler, M. H.; Williams, E. D. (1994) Synthesis and storage in rat thyroid C-cells. J. Histochem. Cytochem. 42 (8), 10551060.
186
CONTENTS
INDEX
R eferences
1.2 rRNA target

A. RNA probe
Cary, S. C.; W arren, W .; Anderson, E.; G iovannoni, S. J. (1993) Identification and localization of bacterial endosymbionts in hydrothermal vent taxa with symbiont-specific polymerase chain reaction amplification and in situ hybridization techniques. Mol. Marine Biol. Biotechn. 2 (1), 5162. Fischer, D.; W eisenberger, D.; Scheer, U.: Assigning functions to nucleolar structures. Chomosoma Focus.
1.3 RNA target

A. O ligonucleotide Probes
b. 3'-Tailing
Negro, F .; Pacchioni, D.; Shimizu, Y .; Miller, R. H.; Bussolati, G .; Purcell, R. H.; Bonino, F . (1992) Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology. Proc. Natl. Acad. Sci. USA 89, 22472251. Azum-G elade, M.-C.; Noaillac-Depeyre, J.; Caizergues-Ferrer, M.; G as, N. (1994) Cell cycle redistribution of U3 snRNA and fibrillarin. J. Cell Sci. 107, 463475.
B. O ligonucleotide probes
a. 3'-Endlabeling
Dixon, B. (1992) Targeting ribosomal RNA: Identifying microbes. BioTechn. 10, 124. Zarda, B.; Amann, R.; W allner, G .; Schleifer, K.-H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted oligonucleoen. Microbiol. 137, 28232830. tides. J. G
B. RNA probes
Bashir, R.; McManus, B.; Cunningham, C.; W eisenburger, D.; Hochberg, F . (1994) Detection of Eber-1 RNA in primary brain lymphomas in immunocompetent and immunocompromised patients. J. Neuro-Oncol. 20, 4753. Di G iuseppe, J. A.; Wu, T .-C.; Corio, R. L. (1994) Analysis of epstein-barr virus-encoded small RNA 1 expression in benign lymphoepithelial salivary gland lesions. Mod. Pathol. 7 (5), 555559. Lima, M. I.; Fonseca, M. E. N.; Flores, R.; Kitajima, E. W . (1994) Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. Arch. Virol. 138, 385390. Lin, N.-S.; Chen, C.-C.; Hsu, Y .-H. (1993) Postembedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections. J. Histochem. Cytochem. 41 (10), 15131519.
b. 3'-Tailing
Azum-G lade, M.-C.; Noaillac-Depeyre, J.; Caizergues-Ferrer, M.; G as, N. (1994) Cell cycle redistribution of U3 snRNA and fibrillarin. Presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase. J. Cell Sci. 107, 463475. Concha, I. I.; Urzua, U.; Y anez, A.; Schroeder, R.; Pessot, C.; Burzio, L. O. (1993) U1 and U2 snRNA are localized in the sperm nucleus. Experiment. Cell Res. 204, 378381.
G ersdorf, H.; Pelz, K.; G bel, U. B. (1993) Fluorescence in situ hybridization for direct visualization of G ram-negative anaerobes in subgingival plaque samples. FEMS Immunol. & Medical Microbiol. 6, 109114. Roller, C.; W agner, M.; Amann, R.; Ludwig, W .; Schleifer, K.-H. (1994) In situ probing of G rampositive bacteria with high DNA G +C content using 23S rRNA-targeted oligonucleotides. Microbiol. 140, 28492858. W allner, G .; Amann, R.; Beisker, W . (1993) Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytom. 14, 136143. Zarda, B.; Amann, R.; W allner, G .; Schleifer, K.-H. (1991) Identification of single bacterial cells using digoxigenin-labeled rRNA-targeted oligonucleotides. J. G en. Microbiol. 137, 28232830.
C. DNA probes
d. PCR
Tanaka, Y .; Enomoto, N.; Kojima, S.; Tang, L.; G oto, M.; Marumo, F .; Sato, C. (1993) Detection of hepatitis C virus RNA in the liver by in situ hybridization. Liver 13, 203208.
CONTENTS
INDEX
187
R eferences
2. DNA target
A. DNA probes
a. RPL
Boland, N. I.; G osden, R. G . (1994) Clonal analysis of chimaeric mouse ovaries using DNA in situ hybridization. J. Reprod. Fertility 100, 203210. Brown, D. W .; W elsh, R. M.; Like, A. A. (1993) Infection of peripancreatic lymph nodes but not islets precedes kilham rat virus-induced diabetes in BB/W or rats. J. Virol. 67 (10), 58735878. Burnett, S.; Kiessling, U.; Pettersson, U. (1989) Loss of bovine papillomavirus DNA replication control in growth-arrested transformed cells. J. Virol. 63 (5), 22152225. Cenacchi, G .; Musiani, M.; G entilomi, G .; Righi, S.; Zerbini, M.; Chandler, J. G .; Scala, C.; La Plaga, M.; Martinelli, G . N. (1993) In situ hybridization at the ultrastructural level: localization of cytomegalovirus DNA using digoxigenin labeled probes. J. Submicrosc. Cytol. Pathol. 25 (3), 341345. Clark, J.; Moore, L.; Krasinskas, A.; W ay, J.; Battey, J.; Tamkun, J.; Kahn, R. A. (1993) Selective amplification of additional members of the ADPribosylation factor (ARF) family: Cloning of additional human and drosophila ARF-like genes. Proc. Natl. Acad. Sci. USA 90, 89528956. Coates, P . J.; DArdenne, A. J.; Slavin, G .; Kingston, J. E.; Malpas, J. S. (1993) Detection of Epstein-Barr virus in reed-sternberg cells of hodgkins disease arising in children. Med. Pediatric Oncol. 21, 1923. Delvenne, P .; Fontaine, M.-A.; Delvenne, C.; Nikkels, A.; Boniver, J. (1994) Detection of human papillomaviruses in paraffin-embedded biopsies of cervical intraepithelial lesions: analysis by immunohistochemistry, in situ hybridization, and the polymerase chain reaction. Modern Pathol. 7 (1), 113119. G entilomi, G .; Musiani, M.; Zerbini, M.; G allinella, G .; G ibellini, D.; La Placa, M. (1989) A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes. J. Immunol. Methods 125, 177183. G enitilomi, G .; Musiani, M.; Zerbini, M.; G ibellini, D.; G allinella, G .; Venturoli, S. (1992) Double in situ hybridization for detection of herpes simplex virus and cytomegalovirus DNA using non-radioactive probes. J. Histochem. Cytochem. 40 (3), 421425. G entilomi, G .; Zerbini, M.; Musiani, M.; G allinella, G .; G ibellini, D.; Venturoli, S.; Re, M. C.; Pileri, S.; Finelli, C.; La Placa, M. (1993) In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labeled probe. Mol. Cell. Probes 7, 1924. Han, K.-H.; Hollinger, F . B.; Noonan, C. A.; Y offe, B. (1992) Simultaneous detection of HBV-specific antigens and DNA in paraffin-embedded liver tissue by immunohisto-chemistry and in situ hybridization using a digoxigenin-labeled probe. J. Virol. Meth. 37, 8998. Heiles, H. B. J.; G enersch, E.; Kessler, C.; Neumann, R.; Eggers, H. J. (1988) In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells. BioTechn. 6, 978981. Heino, P .; Hukkanen, V .; Arstila, P . (1989) Detection of human papilloma virus (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes. J. Virol. Methods 26, 331338. Holm, R.; Karlsen, F .; Nesland, J. M. (1992) In situ hybridization with nonisotopic probes using different detection systems. Modern Pathol. 5 (3), 315319. Han, K. H.; Hollinger, F . B.; Noonan, C. A.; Solomon, H.; Klintmalm, G . B. G .; G enta, R. M.; Y offe, B. (1993) Southern-blot analysis and simultaneous in situ detection of hepatitis B virus-associated DNA and antigens in patients with end-stage liver disease. Hepatol. 18, 10321038. Jackwood, D. J.; Swayne, D. E.; Fisk, R. J. (1992) Detection of infectious bursal disease viruses using in situ hybridization and non-radioactive probes. Avian Diseases 36, 154157. Jacob, J.; Kassir, R.; Kelsoe, G . (1991) In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173, 11651175.
Delvenne, P .; Kaschten, B.; Deneufbourg, J. M.; Demanez, L.; Stevenaert, A.; Reznik, M.; Boniver, J. (1993) Detection of Epstein-Barr virus in a case of undifferentiated nasopharyngeal carcinoma by in situ hybridization with digoxigenin-labeled PCRgenerated probes. Virchows Archiv A. Pathol. Anat. 423, 145150. Fletcher, S.; Darragh, D.; Fan, Y .; G rounds, M. D.; Fischer, C. J.; Beilharz, M. W . (1993) Specific cloning of DNA fragments unique to the dog ATA 10 (34), 7783. Y chromosome. G Furuta, Y .; Inuyama, Y .; Nagashima, K. (1989) Detection of human papillomavirus genome in nasolaryngeal papillomas using digoxigenin labeled DNA probes. J. Otolaryngol. Japan. 92, 20552063. Furuta, Y .; Shinohara, T .; Sano, K.; Meguro, M.; Nagashima, K. (1990) In situ hybridization with digoxigenin-labeled DNA probes for detection of viral genomes. J. Clin. Pathol. 43, 806809.
188
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Kawase, M.; Honda, M.; Niimura, M. (1994) Detection of human papillomavirus type 60 in plantar cysts and verruca plantaris by the in situ hybridization method using digoxigenin labeled probes. J. Dermatol. 21, 709715. Keighren, M.; W est, J. D. (1993) Analysis of cell ploidy in histological sections of mouse tissues by DNA-DNA in situ hybridization with digoxigeninlabeled probes. Histochem. J. 25, 3044. Konkle, B. A.; Shapiro, S. S.; Asch, A. S.; Nachman, R. L. (1990) Cytokine-enhanced expression of glycoprotein Iba in human endothelium. J. Biol. Chem. 265 (32), 1983319838. Lassus, J.; Niemi, K.-M.; Marjamki, A.; Syrjnen, S.; Kartamaa, M.; Lehmus, A.; Krohn, K.; Ranki, A. (1992) Comparison of four in situ hybridization methods, based on digoxigenin- and biotin-labeled probes, in detecting HPV DNA in male condylomata acuminata. Intern. J. STD & AIDS 3, 196203. Lau, J. Y . N.; Naoumov, N. V .; Alexander, G . J. M.; Williams, R. (1991) Rapid detection of hepatitis B virus DNA in liver tissue by in situ hybridization and its combination with immunohistochemistry for simultaneous detection of HBV antigens. J. Clin. Pathol. 44, 905908. Lereuz, M.; Pallier, C.; Vassias, I.; Elouet, J. F .; Romeo, P .; Morinet, F . (1994) Differential transcription, without replication, of non-structural and structural genes of human parvovirus B19 in the UT7/EPO cell line as demonstrated by in situ hybridization. J. G en. Virol. 75, 14751478. Morey, A. L.; Ferguson, D. J. P .; Leslie, K. O.; Taatjes, D. J.; Fleming, K. A. (1993) Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labeled probes. Histochem. J. 25, 421429. Morris, R. G .; Arends, M. J.; Bishop, P . E.; Sizer, K.; Duvall, E.; Bird, C. C. (1990) Sensitivity of digoxigenin- and biotin-labeled probes of detection of human papillomavirus by in situ hybridization. J. Clin. Pathol. 43, 800805. Musiani, M.; G entilomi, G .; Zerbini, M.; G ibellini, D.; G allinella, G .; Pileri, S.; Baglioni, P .; La Placa, M. (1990) In situ detection of cytomegalovirus DNA in biopsies of AIDS patients using a hybridoimmunocytochemical assay. Histochem. 94, 2125. Prelle, A.; Fagiolari, G .; Checcarelli, N.; Moggio, M.; Battistel, A.; Comi, G .P .; Bazzi, P .; Bordoni, A.; Zeviani, M.; Scarlato, G . (1994) Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level. Acta Neuropathol. 87, 371376. Permeen, A. M. Y .; Sam, C. K.; Pathmanathan, R.; Prasad, U.; W olf, H. (1990) Detection of Epstein Barr Virus DNA in nasopharyngeal carcinoma using a non-radioactive digoxigenin-labeled probe. J. Virol. Meth. 27 (3), 261268.
Premoli-de-Percoco, G .; G alindo, I.; Ramirez, J. L. (1992) In situ hybridization with digoxigenin-labeled DNA probes for the detection of human papillomavirus-induced focal epithelial hyperplasia among Venezuelans. Virchows Archiv A. Pathol. Anat. 420, 295300. Schmidt, P .; Meyer, H.; Hbert, P .; Hafner, A.; Andiel, E.; G rabner, A.; Dahme, E. (1994) In situ hybridization for demonstration of equine herpesvirus type 1 DNA in paraffin wax-embedded tissues and its use in horses with disseminated necrotizing myeloencephalitis. J. Comp. Path. 110, 215225. Schwarz, T .F .; Nerlich, A.; Hottentrger, B.; Jger, G .; Wiest, I.; Kantimm, S.; Roggendorf, H.; Schultz, M.; G loning, K.-P .; Schramm, T .; Holzgreve, W .; Roggendorf, M. (1991) Parvovirus B19 infection of the fetus. Histology and in situ hybridization. Am. J. Clin. Pathol. 96 (1), 121126. Schwarz, T .F .; Nerlich, A.; Hillemanns, P . (1993) Detection of parvovirus B19 in fetal autopsies. Arch. G ynecol. Obstet. 253, 207213. Smith, K. L.; Robbins, P . D.; Dawkins, H. J. S.; Papadimitriou, J. M.; Redmond, S. L.; Carrello, S.; Harvey, J. M.; Sterrett, G .F . (1994) c-erbB-2 amplification in breast cancer: Detection in formalin-fixed, paraffin-embedded tissue by in situ hybridization. Hum. Pathol. 25, 413418. Thompson, C. H.; Biggs, I. M.; De Zwart-Steffe, R. T. (1990) Detection of molluscum contagiosum virus DNA by in situ hybridization. Pathol. 22, 181186.
b. Nick translation
Coates, P . J.; Mak, W .P .; Slavin, G .; DArdenne, A. J. (1991) Detection of single copies of Epstein-Barr virus in paraffin wax sections by non-radioactive in situ hybridization. J. Clin. Pathol. 44, 487491. Coates, P . J.; Slavin, G .; DArdenne, A. J. (1991) Persistence of epstein-barr virus in reed-sternberg cells throughout the course of hodgkins disease. J. Pathol. 164, 291297. Cooper, K.; Herrington, C. S.; G raham, A. K.; Evans, M. F .; McG ee, J. OD (1991) In situ human papillomavirus (HPV) genotyping of cervical intraepithelial neoplasia in South African and British patients: Evidence for putative HPV integration in vivo. J. Clin. Pathol. 44, 400405. Cooper, K.; Herrington, C. S.; Lo, E. S.-F .; Evans, M. F .; McG ee, J. O. (1992) Integration of human papillomavirus types 16 and 18 in cervical adenocarcinoma. J. Clin. Pathol. 45, 382384. Del Buono, R.; Fleming, K. A.; Morey, A. L.; Hall, P . A.; Wright, N. A. (1992) A nude mouse xenograft model of fetal intestine development and differentiation. Development 114, 6773. Egawa, K.; Shibasaki, Y .; De Villiers, E.-M. (1993) Double infection with human papillomavirus 1 and human papillomavirus 63 in single cells of a lesion displaying only an human papillomavirus 63-induced cytopathogenic effect. Lab. Invest. 69 (5), 583588.
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189
R eferences
Hegele, R. G .; Bicknell, S. G .; Bailey, D. J.; Cameron, R. G . (1994) In situ hybridization for the Y chromosome reveals a donor origin for a posttransplant lymphoproliferative disorder in a sex-mismatched hepatic allograft. Arch. Pathol. Lab. Med. 118, 795796. Heiskanen, M.; Karhu, R.; Hellsten, E.; Peltonen, L.; Kallioniemi, O. P .; Palotie, A. (1994) High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agaroseembedded cells. BioTechn. 17 (5), 928933. Herrington, C. S.; Burns, J.; G raham, A. K.; Bhatt, B.; McG ee, J. O. (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes. II. Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei. J. Clin. Pathol. (Lond) 42 (6), 601606. Herrington, C. S.; Burns, J.; G raham, A. K.; Evans, M.; McG ee, J. O. (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes. I. Relative sensitivity of both reporter molecules for detection of HPV16 in caski cells. J. Clin. Pathol. (Lond) 42 (6), 592600. Herrington, C. S.; Bhatt, B.; Burns, J.; G raham, A. K.; McG ee, J. O . (1989) Simultaneous non-isotopic in situ hybridization using biotinylated and digoxigeninlabeled probes. J. Pathol. (Edin) 157, 163. Herrington, C. S.; De Angelis, M.; Evans, M. F .; Troncone, G .; McG ee, J. O. (1992) Detection of high risk human papillomavirus in routine cervical smears: Strategy for screening. J. Clin. Pathol. 45, 385390. Herrington, C. S.; G raham, A. K.; Burns, J.; McG ee, J. O. (1989) Enhancement of sensitivity of digoxigenin labeled probes: the evaluation of a biotin independent detection system. J. Pathol. (Edin) 158, 337. Herrington, C. S.; G raham, A. K; McG ee, J. O. (1991) Interphase cytogenetics using biotin and digoxigenin labeled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines. J. Clin. Pathol. 44 (1), 3338. Kerstens, H. M. J.; Poddighe, P . J.; Hanselaar, A. G . J. M. (1995) A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem. 43 (4), 347352. Kerstens, H. M. J.; Poddighe, P . J.; Hanselaar, A. G . J. M. (1994) Double-target in situ hybridization in brightfield microscopy. J. Histochem. Cytochem. 42 (8), 10711077.
Lawrence, J. B.; Marselle, L. M.; Byron, K. S.; Johnson, C. V .; Sullivan, J. L.; Singer, R. H. (1990) Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. 87, 54205424. Leitch, I. J.; Leitch, A. R.; Heslop-Harrison, J. S. (1991) Physical mapping of plant DNA sequences by simultaneous in situ hybridization of two enome 34, differently labeled fluorescent probes. G 329333. Morey, A. L.; Porter, H. J.; Keeling, J. W .; Fleming, K. A. (1992) Non-isotopic in situ hybridization and immunophenotyping of infected cells in the investigation of human fetal parvovirus infection. J. Clin. Pathol. 45, 673678. Scherthan, H. (1991) Characterisation of a tandem repetitive sequence cloned from the deer capreolus capreolus and its chromosomal localisation in two muntjac species. Hereditas (in press). Steilen, H.; Ketter, R.; Romanakis, K.; Zwergel, T .; Unteregger, G .; Bonkhoff, H.; Seitz, G .; Ziegler, M.; Zand, K. D.; Wullich, B. (1994) DNA aneuploidy in prostatic adenocarcinoma: A frequent event as shown by fluroescence in situ DNA hybridization. Hum. Pathol. 25, 13061313. Troncone, G .; Herrington, C. S.; Cooper, K.; De Angelis, M. L.; McG ee, J. O. (1992) Detection of human papillomavirus in matched cervical smears and biopsy specimens by non-isotopic in situ hybridization. J. Clin. Pathol. 45, 308313. W achtler, F .; Schfer, C.; Schedle, A.; Schwarzacher, H. G .; Hartung, M.; Stahl, A.; G onzales, I.; Sylvester, J. (1991) Transcribed and nontranscribed parts of the human ribosomal gene repeat show a similar pattern of distribution in nucleoli. Cytogenet. Cell G enet. 57, 175178. W ood, N. B.; Sheikholeslami, M.; Pool, M.; Coon, J. S. (1994) PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections. Diagn. Mol. Pathol. 3 (3), 200208.
d. PCR
Saeki, K.; Mishima, K.; Horiuchi, K.; Hirota, S.; Nomura, S.; Kitamura, Y .; Aozasa, K. (1993) Detection of low copy numbers of epstein-barr virus by in situ hybridization using nonradioisotopic probes prepared by the polymerase chain reaction. Diagn. Mol. Pathol. 2 (2), 108115. V arma, V . A.; Cerjan, C. M.; Abbott, K. L.; Hunter, S. B. (1994) Non-isotopic in situ hybridization method for mitochondria in oncocytes. J. Histochem. Cytochem. 42 (2), 273276.
190
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B. RNA probes
Brown, C. C.; Meyer, R. F .; G rubman, M. J. (1994) Identification of african horse sickness virus in cell culture using a digoxigenin-labeled RNA probe. J. Vet. Diagn. Invest. 6, 153155. Coen, E. S.; Romero, J. M.; Doyle, S.; Elliott, R.; Murphy, G .; Carpenter, R. (1990) Floricaula: A homeotic gene required for flower development in Antirrhinum majus. Cell 63, 13111322. Marquardt, D. L.; W alker, L. L.; Heinemann, S. (1994) Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells. J. Immunol. 152, 45084515. Su, I.-J.; Chen, R.-L.; Lin, D.-T .; Lin, K.-S.; Chen, C.-C. (1994) Epstein-Barr virus (EBV) infects T lymphocytes in childhood EBV-associated hemophagocytic syndrome in Taiwan. Am. J. Pathol. 144 (6), 12191225.
C. O ligonucleotide probe
b. 3'-Tailing
Cubie, H. A.; Felix, D. H.; Southam, J. C.; Wray, D. (1991) Application of molecular techniques in the rapid diagnosis of EBV-associated oral hairy leukoplakia. J. Oral. Pathol. Med. 20, 271274. Cubie, H. A.; Inglis, J. M.; McG owan, A. M. (1991) Detection of respiratory syncytial virus antigen and nucleic acid in clinical specimens using synthetic oligonucleotides. J. Virol. Meth. 34, 2735. Oraveerakul, K.; Choi, C. S.; Molitor, T .W . (1993) Tissue tropisms of porcine parvovirus in swine. Arch. Virol. 130, 377389. Pacchioni, D.; Papotti, M.; Bonio, F .; Bussolati, G .; Negro, F . (1992) Detection of cytomegalovirus by in situ hybridization using a digoxigenin-tailed oligonucleotide. Liver 12, 257261.
Zischler, H.; Nanda, J.; Schfer, R.; Schmid, M.; Epplen, J. (1989) Digoxigenated oligonucleotide probes specific for simple repeats in DNA enet. fingerprinting and hybridization in situ. Hum. G 82 (3), 227233.
CONTENTS
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191
R eferences
3. Chromosomes/Interphase nuclei
Antonacci, R.; Marzella, R.; Finelli, P .; Lonoce, A.; Forabosco, A.; Archidiacono, N.; Rocchi, M. (1995) A panel of subchromosomal painting libraries representing over 300 regions of the human enet. 68, 2532. genome. Cytogenet. Cell G Arnold, N.; Seibl, R.; Kessler, C.; Wienberg, J. (1992) Non-radioactive in situ hybridization with digoxigenin labeled DNA probes. Biotechnic Histochem. 67 (2), 5967. Arnoldus, E. P . J.; Wiegant, J.; Noordermeer, I. A.; W essels, J. W .; Beerstock, G . C.; G rosveld, G . C.; van der Ploeg, M.; Raap, A. K. (1990) Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet. Cell G enet. 54, 108111. Ashley, T .; Ried, T .; W ard, D. C. (1994) Detection of nondisjunction and recombination in meiotic and , Tp(Y)1Ct] mice postmeiotic cells from XYSxr [XY using multicolor fluorescence in situ hybridization. Proc. Natl. Acad. Sci. USA 91, 524528. Avarello, R.; Pedicini, A.; Caiulo, A.; Zuffardi, O.; Fraccaro, M. (1992) Evidence for an ancestral alphoid domain on the long arm of human enet 89, 247249. chromosome 2. Hum. G Balazs, M.; Mayall, B. H.; W aldmann, F . M. (1991) Simultaneous analysis of chromosomal aneusomy and 5-bromodeoxyuridine incorporation in MCF-7 enet. Cytogenet. 57, breast tumor cell line. Cancer G 93102. Boggs, B. A.; Chinault, C. (1994) Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. USA 91, 60836087. Boschman, G . A.; Buys, C. H. C. M.; Van der Veen, A. Y .; Rens, W .; Osinga, J.; Slater, R. M.; Aten, J. A. (1993) Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro, and in situ hybridization of the amplified product. G enes Chrom. Cancer 6, 1016. Celeda, D.; Aldinger, K.; Haar, F .-M.; Hausmann, M.; Durm, M.; Ludwig, H.; Cremer, C. (1994) Rapid fluorescence in situ hybridization with repetitive DNA probes: Quantification by digital image analysis. Cytometry 17, 1325. Chevillard, C.; Le Paslier, D.; Passage, E.; Ougen, P .; Billault, A.; Boyer, S.; Mazan, S.; Bachellerie, J. P .; Vignal, A.; Cohen, D.; Fontes, M. (1993) Relationship between Charcot-Marie-Tooth 1A and Smith-Magenis regions. snU3 may be a candidate gene for the Smith-Magenis syndrome. Hum. Mol. G en. 2 (8), 12351243. Christmann, A.; Lagoda, P . J. L.; Zang, K. D. (1991) Non-radioactive in situ hybridization pattern of the M13 minisatellite sequences on human metaphase enet. 86, 487490. chromosomes. Hum. G Couturier, J.; G arau-Sastre, X.; SchneiderMaunoury, S.; Labib, A.; Orth, G . (1991) Integration of papillomavirus DNA near myc genes in genital carcinomas and its consequences for protooncogene expression. J. Virol. Vol. 65, No. 8, 45344538. Dal Cin, P .; Aly, M. S.; Delabie, J.; Ceuppens, J. L.; Van G ool, S.; Van Damme, B.; Baert, L.; Van Poppel, H.; Van Den Berghe, H. (1992) Trisomy 7 and trisomy 10 characterize subpopulations of tumorinfiltrating lymphocytes in kidney tumors and in the surrounding kidney tissue. Proc. Natl. Acad. Sci. USA 89, 97449748. Dauwerse, J. G .; Wiegant, J.; Raap, A. K.; Breuning, M. H.; Ommen, van, G . J. B. (1992) Multiple colors by fluorescence in situ hybridization using ratiolabeled DNA probes create a molecular karyotype. Hum. Mol. G en. 1 (8), 593598. Davies, A. F .; Barber, L.; Murer-Orlando, M.; Bobrow, M.; Adinolfi, M. (1994) FISH detection of trisomy 21 in interphase by the simultaneous use of two differentially labeled cosmid contigs. J. Med. G enet. 31, 679685. De Frutos, R.; Kimura, K.; Peterson, K. R. (1990) In situ hybridization of Drosophila polytene chromosomes with digoxigenin-dUTP labeled probes. Methods Mol. Cell. Biol. 2, 3236. Delhanty, J. D. A.; G riffin, D. K.; Handyside, A. H.; Harper, J.; Atkinson, G . H. G .; Pieters, M. H. E. C.; Winston, R. M. L. (1993) Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in situ hybridzation (FISH). Hum. Mol. G en. 2 (8), 11831185. De Leeuw, B.; Suijkerbuijk, R. F .; Balemans, M.; Sinke, R. J.; De Jong, B.; Molenaar, W . M.; Meloni, A. M.; Sandberg, A. A.; G eraghty, M.; Hofker, M.; Ropers, H. H.; G eurts van Kessel, A. (1993) Sublocalization of the synovial sarcoma-associated t(X;18) chromosomal breakpoint in Xp11.2 using cosmid cloning and fluorescence in situ hybridization. Oncogene 8, 14571463.
Boyle, A. L.; Feltquite, D. M.; Dracopoli, N. C.; Housman, D. E.; W ard, D. C. (1992) Rapid physical mapping of cloned DNA on banded mouse chromosomes by fluorescence in situ hybridization. G enomics 12, 106115. Brandt, C. A.; Hindkjaer, J.; Stromkjaer, H.; Pedersen, S.; Sunde, L.; Kolvraa, S. (1993) Molecular cytogenetics: Applications in clinical ynecol. Reprod. genetics. Europ. J. Obstetrics & G Biol. 50, 235242. Brandt, C. A.; Lyngbye, T .; Pedersen, S.; Bolund, L.; Friedrich, U. (1994) Value of chromosome painting in determining the chromosomal outcome in offspring of a 12; 16 translocation carrier. J. Med. G enet. 31, 234237. Celeda, D.; Bettag, U.; Cremer, C. (1992) PCR Amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization. BioTechn. 12 (1), 98102.
192
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4. Primed in Situ Labeling of Nucleic Acids

Abbo, S.; Dunford, R. P .; Miller, T . E.; Reader, S. M.; King, I. P . (1993) Primer-mediated in situ detection of the B-hordein gene cluster on barley chromosome 1H. Proc. Natl. Acad. Sci. USA 90, 1182111824. Brandt, C. A.; Djernes, B.; Stromkjaer, H.; Petersen, M. B.; Pedersen, S.; Hindkjaer, J.; Brinch-Iversen, J.; Bruun-Petersen, G . (1994) Pseudododicentric chromosome 18 diagnosed by chromosome painting and primed in situ labeling (PRINS). J. Med. G enet. 31, 99102. Brandt, C. A.; Kierkegaard, O.; Hindkjaer, J.; Jensen, P . K. A.; Pedersen, S.; Therkelsen, A. J. (1993) Ring chromosome 20 with loss of telomeric sequences enet. 44, detected by multicolour PRINS. Clin. G 2631. Cinti, C.; Santi, S.; Maraldi, N. M. (1993) Localization of single copy gene by PRINS technique. Nucl. Acids Res. 21 (24), 57995800. G osden, J.; Hanratty, D. (1991) Comparison of sensitivity of three haptens in the PRINS (oligonucleotide primed in situ DNA synthesis) reaction. Techniques 3 (4), 159165. G osden, J.; Lawson, D. (1995) Instant PRINS: a rapid method for chromosome identification by detecting repeated sequences in situ. Cytogenet. Cell G enet. 68, 5760. Hindkjaer, J.; Koch, J.; Mogensen, J.; Pedersen, S.; Fischer, H.; Nygaard, M.; Junker, S.; G regersen, N.; Kolvraa, S.; Therkelsen, A. J.; Bolund, L. (1991) Primed in situ labeling of nucleic acids. BFE 8, (12) 752756. Hindkjaer, J.; Koch, J.; Terkelsen, C.; Brandt, C. A.; Kolvraa, S.; Bolund, L. (1994) Fast, sensitive multicolor detection of nucleic acids in situ by PRimed IN Situ labeling (PRINS). Cytogenet. Cell G enet. 66, 152154. Koch, J.; Fischer, H.; Askholm, H.; Hindkjaer, J.; Pedersen, S.; Kolvraa, S.; Bolund, L. (1993) Identification of a supernumerary der(18) chromosome by a rational strategy for the cytogenetic typing of small marker chromosomes enet. with chromosome-specific DNA probes. Clin. G 43, 200203. Koch, J.; Hindkjaer, J.; Kolvraa, S.; Bolund, L. Construction of a panel of chromosome specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cytogen. Cell G en. submitted. Pellestor, F .; G irardet, A.; Andro, B.; Charlieu, J.-P . (1994) A polymorphic alpha satellite sequence for human chromosome 13 detected by oligonucleotide enet. 94, primed in situ labeling (PRINS). Hum. G 346348. Pellestor, F .; G irardet, A.; G enevive, L.; Andro, B.; Charlieu, J. P . (1995) Use of the primed in situ labeling (PRINS) technique for a rapid detection of enet. chromosomes 13, 16, 18, 21, X and Y . Hum. G 95, 1217. Speel, E. J. M.; Lawson, D.; Hopman, A. H. N.; G osden, J. (1995) Multi-PRINS: multiple sequential oligonucleotide primed in situ DNA synthesis reactions label specific chromosomes and produce enet. 95, 2933. bands. Hum. G Terkelsen, C.; Koch, J.; Kolvraa, S.; Hindkjaer, J.; Pedersen, S.; Bolund, L. (1993) Repeated primed in situ labeling: formation and labeling of specific DNA sequences in chromosomes and nucleic. Cytogenet. Cell G enet. 63, 235237. Therkelsen, A. J.; Nielsen, A.; Koch, J.; Hindkjaer, J.; Kolvraa, S. (1995) Staining of human telomeres with primed in situ labeling (PRINS). Cytogenet. Cell G enet. 68, 115118. Volpi, E. V .; Baldini, A. (1993) Multiprins: a method for multicolour primed in situ labeling. Chrom. Res. 1, 257260.
Hindkjaer, J.; Koch, J.; Mogensen, J.; Kolvraa, S.; Bolund, L. (1994) Primed in situ (PRINS) labeling of DNA. Meth. Mol. Biol. 33, 95109.
5. In situ hybridization in suspension

Brown, C. C.; Meyer, R. F .; G rubman, M. J. (1993) Use of digoxigenin-labeled RNA probe to detect all 24 serotypes of bluetongue virus in cell culture. J. Vet. Diagn. Invest. 5, 159162. Timm, A.; Stewart, C. C. (1992) Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry. BioTechn. 12 (3), 362367.
198
CONTENTS
INDEX
R eferences
6. PCR in situ technique

Cinti, C.; Santi, S.; Maraldi, N. M. (1993) Localization of single copy gene by PRINS technique. Nucl. Acids Res. 21 (24), 57995800. G osden, J.; Hanratty, D. (1993) PCR In situ: A rapid alternative to in situ hybridization for mapping short, low copy number sequences without isotopes. BioTechn. 15 (1), 7880. G ressens, P .; Martin, J. R. (1994) In situ polymerase chain reaction: localization of HSV-2 DNA sequences in infections of the nervous system. J. Virol. Meth. 46, 6183. Li, S.; Harris, C. P .; Leong, S. A. (1993) Comparison of fluorescence in situ hybridization and primed in situ labeling methods for detection of single-copy genes in the fungus ustilago maydis. Experim. Mycol. 17, 301308. Long, A. A.; Komminoth, P .; Lee, E.; W olfe, H. J. (1993) Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Histochem. 99, 151162. Moss, R. B.; Kaliner, M. A. (1994) Primed in Situ DNA Amplification (PIDA). J. Clin. Lab. Anal. 8, 120122. Nuovo, G .; Direct incorporation of digoxigenin-11dUTP into amplified DNA using the Hot Start PCR in situ technique. Nuovo, G . J.; G allery, F .; MacConnell, P .; Becker, J.; Bloch, W . (1991) An improved technique for the in situ detection of DNA after Polymerase Chain Reaction amplification. Am. J. Pathol. 139 (6), 12391244. Patel, V .G .; Shum-Siu, A.; Heniford, B. W .; Wieman, T . J.; Hendler, F . J. (1994) Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. Am. J. Pathol. 144 (1), 714. Pellestor, F .; G irardet, A.; Lefort, G .; Andro, B.; Charlieu, J. P . (1994) Dtection rapide des chromosomes 21 in situ par la technqie PRINS. Ann. G nt. 37 (2), 6671. Pestaner, J. P .; Bibbo, M.; Bobroski, L.; Seshamma, T .; Bagasra, O. (1994) Potential of the in situ polymerase chain reaction in diagnostic cytology. Acta Cytol. 38, 676680. Teyssier, M.; Carosella, E.; G luckman, E.; Kirszenbau, M. (1994) PCR introcellulaire: nouvelle approche de diagnostic tissulaire et cytogenetique. Immunoanal. Biol. Spec. 9, 159164. Tsongalis, G . J.; McPhail, A. H.; Lodge-Rigal, R. D.; Chapman, J. F .; Silverman, L. M. (1994) Localized in situ amplification (LISA): A novel approach to in situ PCR. Clin. Chem. 40 (3), 381384. Zehbe, I.; Hacker, G .W .; Rylander, E.; Sllstrm, J.; Wilander, E. (1992) Detection of single HPV copies in SiHa cells by in situ polymerase chain reaction (in situ PCR) combined with immuno-peroxidase and immunogold-silver staining (IG SS) techniques. Anticanc. Res. 12, 21652168.
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General Introduction to In Situ H ybridization

G eneral Introduction to In Situ Hybridization

G eneral I ntroduction to I n Situ H ybridiz ation

Direct and indirect methods

Guidelines for In Situ H ybridization

G uidelines for In Situ Hybridization

Details of the Technique

Pretreatments of material on the slide

D etails of the Technique

Denaturation of probe and target

D etails of the Technique

D etails of the Technique

D etails of the Technique

D etails of the Technique

Flow diagram for in situ hybridization

Fluorescence microscopy for probes directly labeled with a fluorochrome

D etails of the Technique

Procedures for In Situ H ybridization to Chromosomes , Cells, and Tissue Sections

Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections

W hole mount ISH

II. Denaturation and hybridization

I. Pretreatment of metaphase spreads on slides (optional)

B. For unique probes

3 Prepare a probe stock solution by

III. Single color fluorescent detection with immunological amplification

B1. Digoxigenin-labeled probe, low sensitivity

6 Wash the slides (3 x 5 min) at room

7 Pipette 100 l of antibody 2 working

IV . Multicolor fluorescence in situ hybridization (Multicolor FISH)

N ote: Be careful that the selected antibodies do not cross-react.

listed in Table 1 to the target.

V . Results obtained with human metaphase chromosome spreads

Reagents available from Boehringer Mannheim for these procedures

Biotin-Nick Translation Mix* for in situ probes

1 214 624 1 428 578 1 055 097 236 276

VI. T roubleshooting guide for in situ hybridization on chromosome spreads

I f the probe is larger than 500 nt,

of the slide using a diamond stylus. 5 min (in a C oplin jar).

Incubate slide in pepsin solution

D. If there is a starry background fluorescence, then:

Possible cause Agglutination of fluorochromes coupled to antibodies

Labeled probe molecules are too long

E. If there is a general strong staining of chromosomes and nuclei, then:

C. If there is a milky background fluorescence, then:

co-hybridization (normal chromosomes)

DOP-PCR Telenius et al., 1992 5 CCG ACTCG AGNNNNNN ATG TG G 3

Low stringency PCR (T a = 30 C; 5 cycles) ! frequent priming at multiple sites 5 3

I. Preparation and labeling of DO P-PCR products

Higher stringency PCR (T a = 62 C; 35 cycles) ! specific priming of pre-amplified sequences

Figure 2: Schematic illustration of DOP-PCR. For explanation, see text.

6 Analyze aliquots of the reaction mix

4 C o-precipitate the probe D N As in each

II. Comparative genomic hybridization (CG H)

Figure 3: Comparative genomic

Figure 4: Detection of amplified

Figure 5: Detection of amplified

Reagents available from Boehringer Mannheim for these procedures

The D O P-PCR Master contains:

2 x 1000 l One vial No. 4, 50 l

Biotin-Nick Translation Mix* for in situ probes

Product High Pure PCR Product Purification Kit**

Description Kit for 50 purifications Kit for 250 purifications