Part I: The Staining Process

Chapter 6

Detection Methods
Kenneth Petersen, PhD, MSc
Hans Christian Pedersen, MSc

Detect (n.)
To discover or determine the existence or presence of <something>.
Merriam-Webster Online Dictionary

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Detection Methods | Chapter 6

Chapter 6.1 Introduction

more intense staining. The secondary antibodies used in the

indirect methods are typically raised in goat against either

Immunohistochemistry (IHC) has emerged as a powerful in-

mouse (GaM) or rabbit (GaR) antibodies. With the successful

vestigative tool that can provide supplemental information to

application of IHC methods to formalin-fixed specimens, new

the routine morphological assessment of tissues. The use of

staining methods were rapidly developed including the immu-

IHC to study cellular markers that define specific phenotypes

noperoxidase bridge method (6) and the peroxidase anti-per-

has provided important diagnostic, prognostic, and predic-

oxidase (PAP) complex method (7).

tive information relative to disease status and biology. The

application of antibodies to the molecular study of tissue

Table 6.1 Complexity vs. Sensitivity of Detection Systems.

pathology has required adaptation and refinement of IHC

native proteins, in fixed tissues the preservation of antigen

is variable and unpredictable. Thus, the history of IHC has
evolved so that we today are able to detect proteins in tissue
with great sensitivity, and also provide a semi-quantitative assessment, with the ultimate goal of integrating tissue-based

Polymer
(Doublestain)

4
Number of Steps

solution-based immunoassays that detect relatively abundant

iCSA

techniques, particularly for use in fixed tissues. In contrast to

LSAB/ABC

Indirect

The first staining with an antibody to find an antigen in tissue

Polymer
+ Linker
Polymer

Direct

analysis with proteomic information.

Immunohistochemistry: In the Beginning

CSA II

Sensitivity

LSAB=Labeled streptavidin-biotin; ABC=Avidin-biotin complex; CSA

II=Catalyzed signal amplification II, iCSA = Improved catalyzed signal
amplification.

was reported in 1941, using a fluorescence-labeled antibody

(1). Twentyfive years later, the enzyme horseradish peroxidase
(HRP) together with 3,3-diaminobenzidine (DAB) was used to
study mouse kidneys (2). The following year, an antibody linked

Chapter 6.2 Avidin-Biotin Immunohistochemistry

to HRP was used to visualize antigens in tissue using the indirect method, where a second antibody is used to recognize the

The next generation of IHC methods emerged in 1981 with the

first or primary antibody which is attached to the antigen (Figure

avidin-biotin-based methods (Figure 6.2) (8). These methods

6.1). The secondary antibody recognize the constant part (Fc)

are still used to a limited degree in some pathology laborato-

of the primary antibody, which makes it possible to recognize all

ries and rely on the strong affinity of avidin or streptavidin for

primary antibodies as long as they are from the same species.

the vitamin biotin.

These pioneering studies using enzyme labels instead of

fluorescent dyes and the application to formalin-fixed, paraf-

Streptavidin (from the bacteria Streptomyces avidinii) and avi-

fin-embedded tissue (FFPE) (3) opened the door to the use of

din (from chicken egg) both have four binding sites for bio-

immunoperoxidase methods for routine diagnosis in anatomic

tin. The biotin molecule is easily conjugated to antibodies and

pathology (4, 5), and led to the development of modern meth-

enzymes. In the avidin-biotin complex (ABC) method second-

ods of IHC (see Chapter 1).

ary antibodies are conjugated to biotin and function as links

between tissue-bound primary antibodies and an avidin-bio-

The good preservation of features and improved morphology

tin-peroxidase complex. The four binding sites for biotin make

of FFPE of tissues, makes this method the preferred choice in

lattice complexes possible, where the avidins are linked to-

almost every clinical pathology laboratory. The indirect staining

gether via the enzyme (8). The only requirement is that the en-

methods are likewise the preferred staining methods because

zyme has at least two biotin molecules attached so that it can

labeling of the primary antibody is avoided, and they give a

function as a link between the avidins. A colorless substrate,

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Chapter 6 | Detection Methods

e.g. DAB, is subsequently added, and is converted to a brown

Enzyme
Label

Secondary
antibody

end-product by the multiple peroxidase enzyme molecules

now attached at the site of the target antigen.
In a similar method the labeled streptavidin-biotin (LSAB) method also utilizes a biotinylated secondary antibody that links pri-

Primary
antibody

mary antibodies to a streptavidin-peroxidase conjugate (Figure

6.3). This approach has the advantage that preassembly of the

Tissue
antigen
Direct staining

Indirect staining

ABC complex is not needed. In both methods a single primary

antibody is subsequently associated with multiple peroxidase
molecules, and because of the large enzyme-to-antibody ratio,

Figure 6.1 Direct vs. indirect staining.

a considerable increase in sensitivity is achieved compared to

direct peroxidase-conjugate methods.
When using these methods it is important to be aware of their
limitations. Avidin has a tendency to bind non-specifically to

Avidin-biotin enzyme complex

Must be prepared 30 minutes prior to use

lectin-like and negatively charged tissue components at physiological pH. For streptavidin less non-specific tissue binding is
observed. Another challenge is the presence of endogenous biotin in tissues. Formalin fixation and paraffin embedding has been

Biotinylated
secondary antibody

Primary antibody
Tissue antigen

shown to significantly reduce the level of endogenous biotin, but

residual activity can still be observed in tissues such as liver and
kidney. Methods to block endogenous biotin are partially effective, but add another layer of complexity to an already complex
procedure. In frozen tissue sections, the level of endogenous biotin is usually even higher than that encountered in FFPE specimens, giving troublesome non-specific binding of the avidins.

Figure 6.2 Avidin-Biotin Complex (ABC) method.

Chapter 6.3 Polymer-Based

Immunohistochemistry
Streptavidin enzyme complex

Biotinylated
secondary antibody, mouse/rabbit

The limitations associated with the avidin-biotin system, led to

the development of detection systems with higher sensitivity
and specificity, employing polymer-based IHC techniques (9).
These methods utilize a polymer backbone to which multiple
antibodies and enzyme molecules are conjugated. As many

Primary antibody

as 70 enzyme molecules and about 10 primary antibodies

Tissue antigen

can be conjugated to a single dextran backbone. This construct allowed the entire IHC staining procedure, from primary antibody to enzyme, to be accomplished in a single step

Figure 6.3 Labeled Streptavidin-Biotin (LSAB) Method

(10). On the other hand, one limitation of this method was its
restriction to a select group of primary antibodies provided

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Detection Methods | Chapter 6

by the manufacturer, and lack of utility for many user-supplied

cule. The amine can be used to add biotin, or other molecules of

primary antibodies.

interest, to tyramine through an amide bond, hence the tyramide

amplification name also used for this method. When tyramide

To overcome this limitation a new type of visualisation system,

is oxidized, it will react rapidly with electron-rich aromatic com-

EnVision, was introduced (Figure 6.4). This indirect visualiza-

pounds, such as the amino acid tyrosine found in protein mole-

tion system also contains a dextran backbone to which multi-

cules (13). This reaction can be used in IHC to bind biotinyl-tyr-

ple enzyme molecules are attached. However, the EnVision

amide to protein molecules in the immediate vicinity of peroxidase

system contains secondary antibodies with anti-mouse Ig and

enzymes. This reaction results in the deposition of numerous bio-

anti-rabbit Ig specificity. This universal reagent could be used

tin signals around the primary antibody.

to detect any tissue-bound primary antibody of mouse or rabbit

origin. The broad applicability of this method opened the door

In a typical CSA-based IHC procedure, peroxidase en-

to a new family of polymer-based IHC methods. The sensitivity

zymes are first associated with primary antibodies by any

of these methods when compared to LSAB and ABC methods

of the standard IHC methods (Figure 6.5). Biotinyl tyramide

is comparable or even slightly greater in most cases (11). By

and hydrogen peroxide are applied as a substrate to gen-

adding an additional linker step, the sensitivity can be improved

erate numerous biotin signals. These biotin molecules can

further. However, because of the large molecular size of the

then be used to capture subsequent streptavidin-peroxi-

polymer conjugates, accessibility to certain epitopes can be a

dase enzymes to produce the desired staining by addition

challenge, presumably due to steric hindrance.

of the appropriate substrate (14). Another possibility is repetition of the biotinyl-tyramide reaction, which will increase
the numerous biotin signals even further. This cycling of the

Chapter 6.4 Catalysed Signal Amplification (CSA)

reaction is practically limited to two or three cycles before

background staining becomes too high. CSA is a highly sen-

This amplification technique is based on the ability of peroxidase

sitive amplification technique, but has several disadvantag-

enzyme to oxidize phenolic compounds to highly reactive and

es that prevent its general use. The method is time consum-

unstable intermediates called radicals (12). The commonly used

ing, results can be hard to reproduce, and as in previous

substrate in this technique is tyramine. It has a phenol in one end,

biotin-based methods endogenous biotin can give a high

used by peroxidases, and an amine in the other end of the mole-

background staining.

Dextran backbone
Avidin-biotin enzyme complex

HRP enzyme

Secondary antibody, mouse/rabbit

Secondary antibody
Primary antibody
Tissue
antigen

Primary antibody
HRP conjugated
streptavidin

Tissue
antigen

Biotinyl-tyramide
deposition

Figure 6.4 Two-step polymer method (EnVision).

Figure 6.5 The CSA system.

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Chapter 6 | Detection Methods

Chapter 6.5 Fluorescyl-tyramide Amplification

Chapter 6.6 Improved Catalysed Signal

Amplification (iCSA)

Fluorescyl-tyramide can replace biotinyl-tyramide to avoid endogenous biotin background. In this procedure peroxidase is

The latest improvement of the CSA method to increase sensi-

associated with a tissue-bound primary antibody by applica-

tivity and improve signal to noise ratio introduces a new more

tion of a secondary anti-mouse antibody to which peroxidase

soluble substrate. It entails a background-reducing effect,

has been conjugated. The peroxidase catalyzes the conver-

combined with a crosslinker that enhances the precipitation

sion and deposition of fluorescyl-tyramide onto the tissue sec-

of the substrate in step 3 (Figure 6.6) The fluorescein is con-

tion. At this point the reaction can be terminated and viewed

served in the substrate while the tyramine is substituted with

by fluorescence microscopy, or the signal can be converted to

ferulic acid, which is a much better peroxidase substrate. To-

a colorimetric reaction by the sequential application of an an-

gether these changes improve CSA method by maintaining

ti-fluorsecein antibody conjugated to peroxidase followed by a

high sensitivity and reducing background, giving high sig-

diaminobenzidine-hydrogen peroxide substrate.

nal-to-noise ratio. Furthermore, the incubation time in each

step can be reduced significantly making it possible to stain

In comparison to standard IHC methods, tyramide amplifica-

a tissue in less than one hour.

tion methods have typically increased sensitivity by at least 50fold or greater (15). As with any amplification method, background tends to increase along with signal.

Dextran backbone

Substrate and
cross-linker

Enzyme

Primary antibody

Secondary antibody

Tissue
antigen

STEP 1

STEP 2

STEP 3

Red
ed chromogen
ch
AP conjugated
F(Ab) antibody

STEP 4

STEP
P5

Figure 6.6 Improved CSA system (iCSA). A proprietary methodology developed by Dako.

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Detection Methods | Chapter 6

Chapter 6.7 Multi-Staining Immunohistochemistry

expressed in benign prostate tissue. Thus it is used as a positive cancer marker, often in a multiplex stain with keratin and

In some cases there is a need for knowledge about the relative

p63 (see example in Figure 6.8). If single stains are done on

localization of targets, which context can only be obtained by vis-

serial sections, interpretation is much more difficult and ambig-

ualizing multiple targets in one slide. In other cases, the material

uous lesions may be absent in adjacent cuts, especially when

available for staining is scarce and there is a need for multiplexing

dealing with small foci, with the result that some malignancies

to retrieve all possible information out of material available.

may remain undiagnosed. In this context, multiple staining protocols significantly improve the ability to distinguish between

Definition of Multi-Staining IHC

benign and malignant lesions. This approach, which reduces

Multiple staining can be defined as the detection of two or

the percentage of ambiguous lesions and the need for addi-

more targets on one slide, thus increasing the information ob-

tional biopsies, is being extended to facilitate recognition of

tained from each slide and reducing turnaround time, com-

other invasive cancers, as in breast.

pared to single staining or sequential staining (see definition

below). This technique also makes it possible to assess the

Technical Challenges

topographic relationship of two or more targets, for exam-

Before embarking on a multi-staining project, some important

ple, to determine whether targets are present in different cell

issues should be considered:

populations, in different cells, in the same cell, or even in the

Most primary antibodies used today originate from either

same cellular compartment. In addition, multiple staining al-

mouse or rabbit and are visualized using systems based

lows the combination of in situ hybridization (ISH) and IHC,

on anti-mouse and anti-rabbit secondary antibodies. The

giving information about a particular target both at protein

challenge of distinguishing between two primary anti-

level and DNA/mRNA level. Information can also be obtained

bodies of the same species (mouse-mouse, or rabbit-

on possible cell-to-cell spatial contacts of different cell types.

rabbit) must be addressed, because separate mouse and

Furthermore, with an increasing demand for less invasive

rabbit primary antibodies to the chosen targets often are

sampling techniques and smaller and fewer specimens avail-

not available. Utilizing two primary antibodies of the same

able, multiple staining has the advantage not only of conserv-

species can require quite elaborate protocols.

ing tissue, but also saving time and reagents.

Spectral differentiation of stain colors may be difficult,

especially if the targets are co-localized leading to a mix-

Examples of Multiple Staining

ture of colors (18). The mixed color should contrast well

The diagnosis of prostatic intra-epithelial neoplasia (PIN) is

with the two basic colors. In the case where a rare target

just one example of the clinical importance of multiple staining.

is co-localized, the color reaction of the more abundant

Prostate needle biopsy is the preferred method for diagnos-

target will tend to dominate the other.

ing early prostate cancer, but in some cases the diagnosis is

Even if targets are not co-localized it is difficult to balance

uncertain because the biopsy includes only a few malignant

signals so as to enable visualization of a rare target in

glands, or a few hyperplastic or dysplastic glands that are

the same slide as highly expressed targets. An adjustment

difficult to distinguish from cancer (16, 17). Since basal cells

in concentration of the primary antibodies may solve this

typically are present in hyperplastic, and dysplastic glands, as

problem.

well as around in situ (PIN) lesions, but absent in malignant in-

If different targets are viewed under different magni-

vasive glands, the demonstration of basal cells can be used to

fications, it may be difficult to obtain the desired

assist recognition, or exclusion, of invasive cancer. Basal cells

topographic information.

are labeled using high molecular weight cytokeratin, cytokera-

Image analysis approaches, such as spectral separation,

tin (e.g. CK5/6 - cytoplasmic) or p63 (nuclear) immunostaining,

are generally superior to the human eye in segregating the

or both. In addition, AMACR/P504S, is expressed in a high per-

different color reactions in a multiplex stained slide.

centage of prostate carcinomas, but is negative or only weakly

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Chapter 6 | Detection Methods

Pre-treatment

double staining due to incomplete elution of unreacted rea-

Multiple staining, like single staining, can be performed on

gents from the first staining sequence, before application of

any of FFPE tissue sections, frozen sections, cell smears and

the next reagents.

cytospin preparations. Multiple staining may be constrained

by the fact that it may not be possible to find a single tissue
pre-treatment (retrieval) protocol that is optimal for all targets.
In this case, it may be necessary to determine a method that
allows all targets to be stained, although the method may be
sub-optimal for some targets.

Multi-Staining Method Selection

To ensure success, IHC staining using multiple antibodies must
be carefully planned. If primary antibodies of the desired specificity for the two (or more) targets are commercially available,
and made in different species, then there are several different
staining methods that one can choose. However, very often
the choice may be limited by the reagents available (19). Care
must be taken to avoid cross-reactivity between reagents; in
the event that avoidance is not possible, then measures must
be taken to minimize the risk, including additional controls to
detect significant cross reactivity if present.

Figure 6.7 Sequential double staining method performed with the EnVisionTM G|2 Doublestain Kit using polyclonal anti-kappa light chains (red)
and polyclonal anti-lambda light chains (brown) as primary antibodies.
FFPE tissue sections from tonsils.

In general, staining methods can be divided into the following

Elution may become an issue with some high-affinity primary

classes:

antibodies, as these may remain at their binding-site, leading

to spurious double stained structures. Elution also risks de-

Sequential staining

naturing epitopes of antigens to be visualized subsequently.

By this method, one staining procedure succeeds another. For

Furthermore, for some chromogens there is a risk that the first

example, the first antibody is applied to the tissue section fol-

chromogen (DAB in particular) may shield other targets. This

lowed by a labeled detection system such as streptavidin-bi-

technique is, therefore, not recommended for evaluation of

otin horseradish peroxidase (HRP), with a chromogen such as

mixed colors at sites of co-localization, because not all reac-

DAB. The second primary antibody is applied only after the

tion products are capable of surviving the rigorous washing

excess DAB is rinsed off, followed by labeling with a streptavi-

required to remove the antibodies. To avoid such problems

din-biotin alkaline phosphatase (AP) detection system and

and blurry staining results, it is recommended to use the most

a colored chromogen. The biggest advantage of sequential

robust dyes such as DAB, Fast Red, AEC and Blue chromog-

staining is that by this procedure problems related to cross-re-

en first, followed by other less robust dyes.

activity are minimized, possibly due to steric interference.

Simultaneous staining

84

A sequential staining is shown in Figure 6.7. Here, the primary

In a simultaneous double stain, the primary antibodies can

and secondary antibodies from the first staining were eluted

be applied simultaneously. The advantage of this method is

before the staining of the next target was performed. The dis-

that it is less time-consuming because the reagents can be

advantages of sequential staining are: the method cannot be

mixed together. However, the technique can only be used if

used for co-localized targets, the technique often leads to a

the primary antibodies are from different species, or are di-

long staining protocol and carries an inherent risk of incorrect

rectly labeled with different enzymes (20).

Detection Methods | Chapter 6

A simple example of the direct method is when the primary

antibodies are fluorescence-labeled with fluorochromes emitting different colors to allow direct visualization of two or more
targets. This avoids cross-reactivity, but is rarely practical
since some form of amplification is necessary to get sufficient
fluorescent signal. Alternatively, the primary antibodies may
be conjugated directly with enzymes, biotin or haptens, subsequently employing the corresponding secondary antibody
or streptavidin reagent. This approach is less time-consuming
than the sequential method, because primary and secondary
antibodies can be mixed together in two incubation steps.
However, it requires avoiding all cross-reactivity.
With the indirect method it is also possible to apply time-saving antibody cocktails because the primary antibodies are
recognized by different secondary antibodies. Generally, it
is advantageous to use secondary antibodies raised in the

Figure 6.8 Simultaneous double staining performed with EnVisionTM

DuoFLEX using an antibody cocktail containing monoclonal rabbit
anti-AMACR (red), monoclonal mouse anti-HMWCK and monoclonal
mouse anti-CK 5/6 (brown/black). FFPE tissue sections from prostate.

same host in order to prevent any unexpected interspecies

cross-reactivity at the level of the secondary antibody. One

Multi-step staining can be used when the selection of primary

DuoFLEX from

antibodies is limited. However, when using this method it is not

Dako. This system applies a mixture of primary antibodies

possible to mix reagents. Users will often find that the choice

of mouse and rabbit origin, followed by a mixture of the

of staining method is limited by the availability of the primary

secondary goat-anti-mouse and goat-anti-rabbit antibodies

antibodies with respect to species origin or label.

example of such a system is the EnVision

TM

labeled with HRP and AP, respectively. Finally, the chromogens are applied sequentially. The result is a double stain

Difficulties arise when targets are known or suspected to be co-lo-

where the primary mouse antibodies are stained brown with

calized, and the only available primary antibodies are unlabeled

DAB and the primary rabbit antibodies are stained red with

monoclonal mouse antibodies of the same IgG subclass. In that

Permanent Red (for an example, see Figure 6.8). The sys-

case, none of the techniques described above are applicable.

tem has been developed for Dakos line of RTU cocktails of

primary antibodies, but may also be used with other anti-

One solution for such circumstance is the Dako Animal Research

body cocktails or individual antibodies that are sequentially

Kit (ARK), which contains reagents for labeling mouse prima-

incubated on a single slide.

ry antibodies with a biotinylated anti-mouse Fab fragment, followed by blocking of the remaining reagent with normal mouse

Multi-step technique

serum. This approach can be applied to the tissue as part of the

This is an indirect/direct method combining unlabeled primary

multi-step technique (21). The kit uses a non-covalently labeled

antibodies with directly-conjugated antibodies (3). The meth-

antibody, thus avoiding the risk of reducing affinity. In addition,

od starts with staining of the unlabeled antibody/antibodies

only small amounts of primary antibody are needed and the kit

with the appropriate detection system, but without performing

does not require time-consuming purification steps.

the final enzymatic staining reaction. The tissue is blocked

with normal serum from the host of the first primary antibody

Another solution is Zenon Technology (Invitrogen) developed for

before the second, directly-labeled primary antibody is add-

flow cytometry. It essentially uses the same technique and offers la-

ed. The staining ends with the two enzymatic reactions being

beling kits for mouse primary antibodies, available as enzyme con-

performed sequentially.

jugates or conjugated to one of a wide variety of fluorescent dyes.

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Chapter 6 | Detection Methods

Finally, it is important to be aware of the fact that visualization

color, from each individual color. Double staining using chromo-

systems with dual recognition such as the EnVision+ Dual

genic dyes is well-established, but if the targets are co-localized

Link system do not discriminate between species, and thus are

then a percentage of the single colors cannot be easily identified.

only suitable for multiple staining when using the sequential

For triple staining, it is naturally more difficult to choose colors that

method. Visualization kits with amplification layers that are not

can be unambiguously differentiated, and even more so if targets

clearly specified should be avoided, since possible cross-re-

are co-localized. In such cases, a technique known as spectral

activity cannot be predicted.

imaging may be applied (18,19). Spectral imaging allows images

of the single stains to be scanned and by using specialized software algorithms the colors are unmixed, thereby displaying the

Chapter 6.8 Selection of Dyes

distribution and abundance of the individual chromogens.

The primary choice to make when deciding how to make the

Visualizing Low Expressed Targets

targets visible is whether to use immunoenzyme staining or flu-

A narrow dynamic range is a disadvantage for immunoenzymatic

orescence. Both have advantages and disadvantages and in

staining. The precipitation process, which is crucial for this meth-

the end, decisions should be made based on conditions of the

od, is only triggered at a certain threshold concentration of sub-

individual experiment.

strate and product. On the other hand, at high concentrations the

precipitated product may inhibit further reaction. Therefore, it is

Chromogenic Dyes

difficult to visualize rare targets and highly abundant targets in the

Table 6.2 Examples of enzyme/chromogen pairs suitable for triple staining.

same slide. To ease this problem, catalyzed signal amplification

Enzyme

Chromogen

Color

- an extremely sensitive IHC staining procedure can be used (Fig-

Gal

X-Gal

Turquoise

ures 6.5 and 6.6). The method can bring low expressed targets

AP

Fast Blue BB

Blue

HRP

AEC

Red

HRP

DAB

Brown

Gal

X-Gal

Turquoise

AP

Liquid Fast Red

Red

It is equally necessary to select dyes with distinguishable

HRP

DAB

Brown

spectral properties. However, there are more colors availa-

AP

New Fucsin

Red

HRP

TMB

Green

Gal (beta-galactosidase); X-Gal (5-bromo-4-chloro-3-indolyl -galactoside); AP (alkaline phosphatase); HRP (horseradish peroxidase);
AEC (3-amino-9-ethylcarbazole); DAB (3,3-diaminobenzidine); TMB
(3,3,5,5-tetramethylbenzidine)

within the same dynamic range as high expressed targets.

Fluorescent Dyes
Double immunofluorescence labeling is quite well established (22). Some of the same considerations as for chromogenic dyes apply when working with immunofluorescence.

ble and the emissions spectra of the fluorescent molecules

are narrower than the spectra of the chromogenic dyes. It is
possible to have more stains on one slide with fluorescent
dyes than it is with chromogenic dyes, which is one of the
main advantages of fluorescent dyes in multistaining. The
use of multiple fluorescent colors is also well established in
FISH and flow cytometry. When using fluorescence dichroic excitation/emission, filters are employed to separate the

86

When selecting color combinations for multiple staining with chro-

different fluorescent signals. The spectral separation can

mogenic dyes, it is advisable to choose opposing colors in the

be aided by digital compensation for overlapping emission

color spectrum such as red and green to facilitate spectral differ-

spectra. In addition, new fluorescence microscope systems

entiation. If using a counterstain, this color must also be included

can separate the spectral signatures of up to eight fluoro-

in the considerations. When working with co-localized targets,

chromes without any problems, using multi-spectral imaging

dyes must be chosen so that it is possible to distinguish the mixed

techniques such as emission fingerprinting (23).

Detection Methods | Chapter 6

When staining co-localized targets, fluorescent dyes may al-

within the desired range. Visualizing a combination of several

low separate identification of targets. This makes it possible to

gates, with the color selected independently of the dyes used

discern targets even in very different concentrations, whereas

for staining, may clarify pictures and facilitate interpretation.

subtly mixed colors from chromogenic dyes may easily pass

This capability also makes it possible to set a threshold on sig-

unnoticed with immunoenzyme staining.

nal intensity, to exclude non-specific staining or background

staining from final images. A more thorough discussion of im-

Thus immunofluorescence has some advantages, but there

age acquisition and analysis can be found in Chapter 7.

are also inherent problems; mainly loss of morphologic detail,

which may determine the choice technique for a multi-staining application.

Chapter 6.10 Immunofluorescence

Alternative dyes

Immunofluorescence (IF) is a common laboratory technique

Alternatives to the conventional chromogenic dyes are colloidal

used in almost all aspects of biology. This technique, based

gold-labeled antibodies that can be used with bright field micros-

on pioneering work by Coons and Kaplan (26, 27), and later

copy, with silver enhancement, Green Fluorescent Proteins (GFP

by Osborne (28), has been widely both in research and clinical

and their variants), and Quantum dots. The latter, especially, has

diagnostics. Applications include the evaluation of cells in sus-

been found to be superior to traditional organic dyes on several

pension, cultured cells, frozen tissue, FFPE tissue, beads, and

counts, such as brightness (owing to the high-quantum yield), as

microarrays for the detection of specific proteins. In IF tech-

well as their higher stability (owing to less photodestruction). They

niques, antibodies are chemically conjugated to fluorescent

can be linked to antibodies or streptavidin as an alternative to

dyes such as fluorescein isothiocyanate (FITC) or tetramethyl

fluorochromes (24). However, the size of these conjugates poses

rhodamine isothiocyanate (TRITC). As in the enzymatic meth-

problems of steric interference and diffusion, in terms of getting

ods these labeled antibodies can be use directly or indirectly

these inorganic particles into cells or organelles.

to bind to the antigen of interest, which allows for antigen detection through fluorescence techniques. The degree of fluo-

Chapter 6.9 Automated Image Acquisition and

Analysis in Multiple Staining

rescence can then be quantified using a flow cytometer, array

scanner, or automated imaging instrument, or visualized using
fluorescence or confocal microscopy. IF techniques can be
used on both fresh and fixed tissue samples, though the latter

Digital image analysis will increase the number of usable dyes

present problems of autofluorescence.

because it does not rely on the human eye for detection and
differentiation. A digital image is acquired at excitation wavelengths relevant for the dyes applied, and separate detectors re-

Table 6.3 Advantages and disadvantages of direct and indirect immunofluorescence.

cord individual colors. Thus, digital image analysis will allow the
combination of both fluorescent and immunoenzyme dyes (25).

Pros

Detectors, however, have biased color vision. They amplify

colors differently than does the human eye. Therefore, dyes

Direct Immunofluorescence

Indirect Immunofluorescence

Simpler

Higher signal (amplified)

Antibodies from the same

species

Flexibility (array of fluorescent

colored secondary antibodies)

used in image analysis should be optimized for the best fit possible with the detectors filter properties.
Image analysis systems incorporate algorithms that allow

Low costs
Cons

Lower signal

More steps

Higher costs

Antibodies from the same

species cannot be used
together

Less flexibility

Background may be amplified

compensation for overlapping emission spectra, comparable

to flow cytometry. They also allow signal gating within a range
of wavelengths of interest, enabling users to see only signals

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Chapter 6 | Detection Methods

Principle of Fluorescence
Fluorescence and phosphorescence are both types of lu-

Molecule goes into

higher energy state

minescence. When molecules with luminescent properties

absorb light, they emit light of a different wavelength. With
fluorescence the emission of light occurs extremely rapidly
after the absorption of excitation light, whereas with phosphorescence emission continues for milliseconds to minutes after

Excitation decay
energy (heat) lost towards
semi-stable state

High energy
Excitation
by external
light source

Light emission
when the molecule goes
back to ground state

the energy source has been removed. Fluorescent materials

give off light because of their atomic structure. Electrons are
arranged in discrete energy levels surrounding the atoms nucleus, with each level having a predetermined amount of energy. When an electron absorbs the energy from a photon of

Low energy

light (Figure 6.10) it becomes excited and jumps to a higher,

less stable, energy level. The excited state does not last long.

Figure 6.10 Principle of fluorescence.

The half-life of the excited state is generally less than 10 seconds. The electron loses a small amount of energy as heat,
and the remainder of the extra energy is given off in the form

A range of wavelengths of light can excite the electrons of a

of a photon. The emitted fluorescence has a lower energy

fluorochrome. For example, fluorescein will fluoresce when hit

than the absorbed light, so the wavelength of the emitted light

by light with any wavelength between 450 nm and 520 nm.

is longer than that of the excitation light.

However, the closer the excitation wavelength is to 495 nm, the

Figure 6.9 Cultured pulmonary artery endothelial cells stained for tubulin (red), actin (green) and DNA (blue). The dual immunofluorescence procedure used rabbit-anti-actin and mouse-anti-alpha tubulin as primary antibodies. The secondary antibodies used were Texas Red-conjugated
goat, anti-rabbit IgG and FITC-conjugated goat, anti-mouse IgG. The sample was also stained with the DNA-specific dye Hoechst 33342. Scale
bar is equal to 20 microns.

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Detection Methods | Chapter 6

more fluorescence will be produced. This optimal wavelength

lem with immunofluorescence is photobleaching. Photobleaching

is called the excitation peak. Similarly, the light produced by

is when the fluorophore looses its ability to fluoresce. This pho-

fluorochromes has a range of wavelengths. The emission of

tochemical destruction is due to the generation of reactive ox-

light from fluorescein ranges from 490 nm to 630 nm, and the

ygen species in the specimen as a byproduct of fluorescence

emission peak is approximately 515 nm. Since the phenome-

excitation (Figure 6.12). Photobleaching can be minimized by: (a)

non of fluorescence was first explained in 1852 by a British sci-

decreasing the excitation light in both intensity and duration, (b)

entist, Sir George Stokes, the shift in wavelength from short to

reducing the availability of singlet oxygen (1O2) by the addition of

long during fluorescence is called Stokes shift (Figure 6.11).

singlet oxygen scavengers (= antifade reagents), and (c) using a

low concentration of a fluorochrome with high-quantum efficiency.

Some fluorochromes have a small Stokes shift, while other fluorescent compounds have large Stokes shifts. For example,

Autofluorescence

the fluorochrome fluorescein can be excited by blue-green

Biological autofluorescence in mammalian cells due to flavin

light, and its Stokes shift is only about 20 nm, which means

coenzymes (FAD and FMN: absorption, 450 nm; emission, 515

that the light emitted is green. This contrasts with another flu-

nm) and reduced pyridine nucleotides (NADH: absorption, 340

orochrome, phycoerythrin, which also can be excited by blue-

nm; emission, 460 nm) can be problematic in the detection

green light, but has a large Stokes shift and thus the light will

of fluorescence probes in tissues and cells. Fixation with al-

be emitted in a different color (yellow).

dehydes, particularly glutaraldehyde, can result in high levels

of autofluorescence. This can be minimized in fixed cells by

Photobleaching

washing with 0.1% sodium borohydride in phosphate-buff-

As with most fluorescence-based techniques, a significant prob-

ered saline (29) prior to antibody incubation. Problems due to

Excitation source, 470 nm

Peak FITC excitation, 495 nm
Intensity (%)

Stroke
shift

100

Energy
Peak FITC emision, 520 nm

S3

80

S2
Intersystem crossing

60

S1

T1

Actual FITC emision, 520 nm

40

Ground
energy state

S0

20

Absorption (blue)
0
400

Phophoresence

Green fluorescence emission

425

450

475

500

525

550

575

600

625

650

675

700

Wavelength (nm)

Figure 6.11 Excitation and emission spectrum of fluorescein. When fluorescein is excited at a wavelength other than its peak excitation (470
nm in this example), the shape of the emission curve (darker green)
remains the same, but the relative intensity is reduced. The efficiency
of the excitation at 470 nm is 45% of peak excitation.

Figure 6.12 Illustration of how a singlet-excited state can convert to

a triplet-excited state. Photobleaching is the irreversible decomposition of the fluorescent molecules in the exited state because of their
interaction with molecular oxygen prior to emission.

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Chapter 6 | Detection Methods

autofluorescence can be minimized by selecting probes and

small amount of FITC fluorescence within the PE band. These

optical filters that maximize the fluorescence signal relative

unwanted signals must be electronically removed or the meas-

to the autofluorescence. Other factors that limit IF include the

urement for each detector will overestimate the actual signal.

performance of the detection instrument (i.e. how well the mi-

This process is called fluorescence compensation and can be

croscope has been calibrated and set), the specificity of the

automatically calculated in many detection systems using sin-

antibodies, and the specimen preparation.

gle positive controls.

Fluorescence Overlap

Applications of IF in Pathology

One of the problems that must be dealt with when measuring

Some practical applications of IF in diagnostic pathology are:

fluorescence of more than one color is the possibility that the

Analysis of protein antigens in fresh, frozen or, less often,

emission signals overlap. It is necessary to remove the over-

fixed tissues; sub-cellular localization of protein antigens

lapping signal or it will give a false level for one or more colors.

in tissue culture monolayers; and observation of bacterial

For example, as shown in figure 6.14, there is significant over-

or parasitic organisms. Immunofluorescence is primarily

lap when using FITC and PE. A range of wavelengths will be

used in the research setting, or in clinical research setting,

collected for each detection channel. In the figure, these are

on frozen tissue. In particular where antibodies compatible

identified as the FITC detector bandwidth and the PE detector

with formalin fixation and paraffin embedding have not

bandwidth. These band-pass optical filters will allow photons

been developed.

within this wavelength range to reach the detector. However,

A major practical use is for fluorescence in situ hybri-

as can be seen in Figure 6.14, there is a very small amount of

dization (FISH), fluorescent labeled DNA is used to detect

PE fluorescence, which is within the FITC band, and similarly a

gene aberrations in cells.

FITC detector PE detector

bandwidth bandwidth

Intensity (%)
100

Excitation
source

80

60

40
Overlap
20

FITC emision
0
400

425

450

475

PE emision
500

525

550

575

600

Wavelength (nm)

Figure 6.13 NADH autofluorescence in a human colon carcinoma cell line

(HCT116). Ultra-violet excitation at 363 nm was used and the emitted fluorescence greater than 440 nm was collected. Scale bar is 10 microns.
Courtesy of Giselle M. Knudsen, Department of Medicinal Chemistry and
Molecular Pharmacology, Purdue University, West Lafayette, IN, USA.

90

Figure 6.14 Fluorescence overlap of FITC and PE.

625

650

675

700

Detection Methods | Chapter 6

Immunofluorescence potentially has a wider dynamic ran-

ge than immunoenzyme staining, as there is no enzymatic

amplification involved and thus the dynamic range is de-

termined solely by the sensitivity of the detectors (25).

Quantitative immunofluorescence staining coupled with

digital scanning of slides and image analysis algorithms

have been utilized to create an automated quantitative im-

munofluorescence technique which has been applied in

various studies (30).

Multi-staining (see multi-staining section)

Visualization of cell structures by super resolution microscopy

Chapter 6.11 Future Perspectives

The IHC technique continues to undergo evolution and improvement, driven by ongoing demands of reproducibility, sensitivity and quantification. Today, automated systems enable
standardized visualization of targets in tissue with increased
sensitivity and improved signal to background ratio. Chromogen, fluorescence and multistain technologies are being employed. Increasingly, stained slides are submitted for digital
scanning and signals quantified using image analysis algorithms. The demand for more information from each slide, to
conserve available tissue, will inevitably lead to increasing use
of multistaining technologies in the pathology laboratories.
In addition, targeted therapies have created a need for more
quantitative biomarker information, launching a rapidly growing
range of new types of IHC tests, variously termed prognostic
markers, predictive markers, companion diagnostics or advanced personalized diagnostics (Chapter 11). Thus, future
IHC-based tests will increasingly rely upon standardized, approved kits and reagents, in combination with an automated image analysis system for the evolution into quantitative pathology.

Acknowledgements
Sections, in whole or parts thereof, from the previous editions of
this Guidebook are used in the 6th edition. We sincerely thank
and acknowledge the contribution of the authors. Special acknowledgements to: Mark Key, J. Paul Robinson, Jennifer Sturgis,

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