Multiple-labeling

with
Immunofluorescen
ce: Methodological
Considerations
Henry Li
2008-03-31
 Introduction to
Immunohistochemistry
http://www.ihcworld.com/introductio
 Immunohistochemistry is the localization of
antigens or proteins in tissue sections by the use
of labeled antibodies as specific reagents through
antigen-antibody interactions that are visualized
by a marker such as fluorescent dye, enzyme, or
colloidal gold. 
 IntroductionTissue Preparation (Fixation,
Sectioning, Whole Mount Preparation)
 Antigen RetrievalIHC Methods (Blocking,
Controls, Direct Method, Indirect Method, PAP
Method, ABC Method, LSAB Method, Polymeric
Method, CSA Method, Sensitivity Chart)
 Multiple Labeling
 Immuno-Electron Microscopy
Tissue Preparation:- Fixation  

  the preservation of tissue
Tissue preparation is the cornerstone of
immunohistochemistry. To ensure
architecture and cell morphology, prompt and adequate fixation is
essential. However, inappropriate or prolonged fixation may
significantly diminish the antibody binding capability. 
 There is no one universal fixative that is ideal for the
demonstration of all antigens. However, in general, many antigens
can be successfully demonstrated in formalin-fixed paraffin-
embedded tissue sections. The discover and development of
antigen retrieval techniques further enhanced the use of formalin
as routine fixative for immunohistochemistry in many research
laboratories. 
  For best results, vertebrate tissues (especially neuronal tissues)
usually require fixation by transcardial perfusion for optimal
tissue preservation. The most common fixatives used for
immunohistochemistry are the followings:
  a) 4% paraformaldehyde in 0.1M phosphate buffer
 b) 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate
buffer
 c) PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2%
lysine in 0.1M phosphate buffer
 d) 4% paraformaldehyde with 0.05% glutaraldehyde (TEM
immunohistochemistry)
  Sectioning
  Since its introduction, paraffin wax has remained the most widely used
embedding medium for diagnostic histopathology in routine histological
laboratories. Accordingly, the largest proportion of material for
immunohistochemistry is formalin-fixed, paraffin-embedded. Paraffin
sections produce satisfactory results for the demonstration of majority of
tissue antigens with the use of antigen retrieval techniques.  
 Certain cell antigens do not survive routine fixation and paraffin
embedding. So the use of frozen sections still remains essential for the
demonstration of many antigens. However, the disadvantage of frozen
sections includes poor morphology, poor resolution at higher
magnifications, special storage needed, limited
retrospective studies and cutting difficulty over paraffin
sections. 
 Vibratome sections have some advantages when doing
immunohistochemistry since the tissue is not processed through organic
solvents or high heat, which can destroy the antigenicity. In addition, the
morphology of tissue sections is not disrupted due to no freezing and
thawing needed. Vibratome sections are often used for floating
immunostaining, especially for pre-embedding EM immunohistochemistry.
The disadvantage of vibratome sections is that the sectioning process is
slow and difficult with soft and poorly fixed tissues. In addiction, the chatter
marks or vibratome lines are often appeared in the sections.  
 Whole Mount
Preparation
 Small blocks of tissue (less than 5 mm
thick) can be processed as whole mounts.
The advantage of whole mount
preparations is that the results provide
three dimensional information about the
location of antigens without the need for
reconstruction from sections. However, the
major limitation of using whole mounts is
antibody penetration may not be
complete in the tissue, resulting in
uneven staining or false negative staining.
So Triton X-100 or saponin treatment are
used routinely for whole mount
 Antigen Retrieval

 The demonstration of many antigens can be

significantly improved by the pretreatment with
the antigen retrieval reagent that break the
protein cross-links formed by formalin fixation
and thereby uncover hidden antigenic sites. The
techniques involved the application of heat for
varying lengths of time to formalin-fixed, paraffin-
embedded tissue sections in an aqueous solution
(commonly referred to as the retrieval solution).
This is called "Heat Induced Epitope Retrieval
(HIER)". Another method uses enzyme digestion
and is called "Proteolytic Induced Epitope
Retrieval (PIER)". 
 Blocking
 Choice: normal host ( species of
the 2nd antibody) serum with BSA
and other supplement
 Caution:  Never block with normal
serum or IgG from the host species
of the primary antibody when using a
labeled, secondary antibody.
Basic Antibody Structure
 How to Choose the Best Secondary
Antibody for
Immunohistochemistry Application
 Selection of the best secondary antibody can improve immunostaining and reduce
false positive or negative staining. Here is some useful information that will help you
to choose the best secondary antibody possible for your specific
immunohistochemical application.
http://www.ihcworld.com/_technical_tips/2nd_antibody_tips.htm
 Species of Primary Antibody:
  The secondary antibody should be against the species that primary antibody is
raised. For example, if the primary antibody is raised in mouse, an anti-mouse
secondary antibody should be used. If it is raised in rabbit, an anti-rabbit secondary
antibody should be used.
  Class and Subclass of Primary Antibody:
  The secondary antibody should match the class or subclass of the primary antibody
used. This is especially true for monoclonal antibodies. For example, if the primary
antibody is mouse IgM, an anti-mouse IgM or anti-mouse IgG should be used.
  If the primary antibody (monoclonal) is one of the mouse IgG subclasses (IgG1,
IgG2a, IgG2b, IgG2c, IgG3), any of the anti-mouse IgG can be used. If the class and/or
subclass of the primary antibody are not known, the anti-mouse IgG may be used
since they recognize most of mouse IgG subtypes.
  
 One can also use a secondary antibody specific to the IgG subclass of the primary
antibody. For example, if the primary antibody is mouse IgG2a, an anti-mouse IgG2a
secondary antibody can be used and this is especially useful for double labeling
methods.
  
 Polyclonal antibodies (such as rabbit, goat, sheep or donkey) are typically IgG class
immunoglobulins so the anti-IgG secondary antibodies for these species may be
used.
 Adsorbed Secondary
Antibody:
 Some of the secondary antibodies have been adsorbed with
animal or human IgG. These antibodies are designed for
particular applications to reduce non-specific background
staining. For example, if working with rat tissues or cells,
choose a secondary antibody that has been adsorbed with
rat serum or IgG. However, such adsorbed antibodies have
greatly reduced epitope recognition and may recognize
some subclasses of IgG very weakly, especially those
subclasses which are most closely homologous to the
species they were adsorbed against. For example, do not
use an anti-mouse IgG that has been adsorbed against rat
IgG unless you are trying to detect a mouse primary
antibody in rat tissue that contains rat immunoglobulin, or
in some other tissue in the presence of a rat primary
antibody. Conversely, if you wish to detect a mouse primary
antibody in the absence of rat immunoglobulins, it is best to
use an anti-mouse secondary antibody that has not been
adsorbed against rat.
 Whole IgG or F(ab')2
Fragments of the

Secondary Antibody
Anti-IgG (H+L): This antibody reacts with both the heavy
and light chains of the IgG molecule, i.e. it reacts with both
the Fc and F(ab')2 portions of IgG. Anti-IgG (H+L) also
reacts with other immunoglobulin classes (e.g. IgM, IgA,
etc.) since all immunoglobulins share the same light chains
(either kappa or lambda). This is commonly used secondary
antibody.
 Anti-IgG, Fc fragment specific: This antibody reacts with
the Fc portion of the IgG heavy chain. In each case, it has
been adsorbed against F(ab')2 fragments. In some cases,
the antibodies are additionally adsorbed to minimize
possible cross-reactivity to IgM and IgA, or to IgM alone.
 Anti-IgG, F(ab')2 fragment specific: This antibody has
been adsorbed against Fc fragments and therefore reacts
only with the Fab portion of IgG. Since it reacts with light
chains, it also reacts with other immunoglobulins sharing
the same light chains. This antibody is used for specific
applications such as double labeling methods or for tissues
or cells that have Fc receptors (thymus, spleen).
 How to Choose a Secondary
Antibody
 In what animal was the primary antibody developed?

 What is the class and/or subclass of the primary antibody :This is primarily
important in the case of monoclonal antibodies. Polyclonal antibodies (generally
developed in rabbit, goat, sheep or donkey) are typically IgG class immunoglobulins.
For this reason, the secondary antibodies for these species will mainly be anti-IgG. If
the primary monoclonal is one of the mouse IgG subclasses (IgG1, IgG2a, IgG2b,
IgG3), almost any of anti-mouse IgG secondary antibodies should bind to it.
However, different specificities to provide the end-user with options, especially for
specific double labeling experiments. If the class and/or subclass of the primary
antibody is not known, the anti-Mouse IgG (Fab) secondary antibodies may be used
since they recognize most mouse immunoglobulin subtypes.

 What form of antibody should be used?

 What kind of label?

 Is there an appropriate adsorbed secondary antibody available?
 Is a F(ab )2 fragment product necessary?
 If all else is equal, decide based on price/titer and availability.
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Antibody_Explorer/Technical_Support/Secondary_Antibody.html
 Selection and Location of Affinity-
Purified
Antibodies
http://www.jacksonimmuno.com/technical/select
 Step 1 Select either "Whole
Molecules" or "F(ab')2 Fragments" of
the antibody
 Step 2 Find the species of the
antigen with which you would like the
antibody to react
 Step 3 Select the host species in
which you want the antibody to be
made
 Step 4 Select most appropriate
antibody from the multiple choices: to
minimize cross-reactivity
 Step 5 Find the price, size, and code
 Controls
 Immunocytochemistry is used for antibody
localization of proteins in cells and tissues. The
specificity of the results depends on two
independent criteria: the specificity of the
antibody and of the method used. The antibody
specificity is best determined by immunoblot and
or immunoprecipitation. Absorption of the
antibody with a protein does not determine that
the antibody would have bound to the same
protein in the tissue, and therefore is not a good
control for antibody specificity. The specificity of
the method is best determined by both a
negative control, replacing the primary antibody
with serum, and a positive control, using the
antibody with cells known to contain the protein.
With the increasing use of immunocytochemistry,
it is important to be aware of the appropriate
controls needed to show specificity of the labeling
(Burry 2000).
 Controls
 ***Special controls must be run in order to test the protocol and
for the specificity of the antibody being used.
  
 Positive control is to test a protocol or procedure and make sure it
works. It will be ideal to use the tissue of known positive as a control.
If the positive control tissue showed negative staining, the protocol or
procedure needs to be checked until a good positive staining is
obtained.
  
 Negative control is to test for the specificity of an antibody involved.
First, no staining must be shown when omitting primary antibody or
replacing an specific primary antibody with normal serum (must be
the same species as primary antibody). This control is easy to achieve
and can be used routinely in immunohistochemical staining.
  
 Second, the staining must be inhibited by adsorption of a primary
antibody with the purified antigen prior to its use, but not by
adsorption with other related or unrelated antigens. This type of
negative control is ideal and necessary in the characterization and
evaluation of new antibodies but it is sometimes difficult to obtain the
purified antigen, therefore it is rarely used routinely in
immunohistochemical staining. Adsorption controls as minimally
acceptable documentation, however, the power of adsorption test is
 Control for Antibodies
 WHAT IS AN ADEQUATE SET OF CONTROLS
FOR AN ANTIBODY? Does the antibody actually reveal
the antigen of interest, or is the binding an artifact? The gold
standard for deciding this issue is to stain tissue with and without
the antigen of interest.

Repeat the experiment for the purpose of
verification
 DON'T BELIEVE EVERYTHING YOU SEE: it is
important to have sufficient controls to convince a skeptical
observer that the staining pattern observed really does represent
the antigen, and wherever possible the results should be checked
with other techniques. (unless you understand how the
images were captured and processed)
Clifford BS, Sawchenko PE. 2003. Magic peptides,
magic antibodies: Guidelines for appropriate
controls for immunohistochemistry. J Com Neurol
 Introduction to Multiple
 Labeling
It is often useful to be able to stain for two or
more antigens in one common tissue section. This
can be achieved by immunofluorescence
method using different fluorescent dyes.
Multiple staining can also be done with
peroxidase conjugated antibodies developed with
different chromogen substrates to produce the
end products of different colors. There are three
basic approaches in planning multiple staining:
parallel, sequential and adjacent. In addition, the
antibody dilution and condition are also important
factors to be considered.  Finally, appropriate
color combination is also crucial since improper
color combination may produce poor result and
fail to demonstrate multiple antigens in the same
section. For best result, the careful design and
test of multiple staining protocols are necessary.
 Whatever labelling combination is
 Controls for confocal laser
scanning microscope
(CLSM)
Multiple immunofluorescence labeling in
combination with CLSM is a powerful strategy to
visualize the spatial relationship between
different antigens in histological sections.
 Samples:
 Dorsal root ganglia from adult Sprague-Delay rats
 Primary Antibodies:
 p75NTR monoclonal antibody (IgG192, IgG1); Rabbit anti
S100 and rabbit anti-P75NTR (#9991) ployclonal
antibodies.
 Secondary antibodies:
 Donkey anti-mouse IgG(H+L) conjugated to Cy3 (Jackson
Immuno)
 Donkey anti-mouse IgG(H+L) conjugated to Alexa Fluor
488 (probes-Invitrogen)
 Donkey anti-rabbit IgG(H+L) conjugated to Alexa Fluor
488 (probes-Invitrogen)
A. Control for specificity I—positive
control
 Rationale: in every control experiment a separate positive control
check has to be performed in parallel in order to be sure that
there is a positive signal in the appropriate channel in the CLSM in
the first place (a channel is a specific setup of the CLSM including
illumination with one of the lasers and the filter and mirror
settings specific for that particular laser and its corresponding
fluorophore-to-be-detected). It is important to know a priori that
the immunocytochemical reactions have been successfully
completed since otherwise no firm conclusions about the degree
of success of the immunocytochemical procedures can be drawn
when no image is obtained in the CLSM.
 Next to determining the specificity of the primary-secondary
antibody binding, these sections are used to check the specificity
of the parameters of the CLSM: does the right fluorophore produce
a detector signal with the corresponding laser and filter settings,
while it produces no detector signal when illuminated with the
non-corresponding (second) laser seen through the filter settings
belonging to the second laser's setup.
 Incubate sections as follows:
 • mouse anti-p75NTR antibodies followed by incubation with
Donkey anti-mouse IgG conjugated to Cy3;
 • rabbit anti-S100 antibodies followed by incubation with Donkey
anti-rabbit IgG conjugated to 488
 Expected results: labeled cells only in the corresponding channel
 Control for specificity II-non
crossing reaction control
 Rationale: controls to be sure that each of the
primary antibodies does not react with the non-
corresponding secondary antibody-conjugate.
 Incubate sections as follows:
 • mouse anti-p75NTR followed by incubation with
donkey anti-rabbit IgG conjugated to 488;
 • rabbit anti-S100 followed by incubation with
donkey anti-mouse IgG conjugated to cy3.
 Expected results: blank sections. If labeled cells
appear in any of these sections, then the “wrong”
secondary antibody binds to the primary
antibody. It should be noted that these sections
must be observed in the CLSM at a high laser
illumination setting in order to detect even traces
of fluorescence.
 C. Control to monitor IgG
interactions
 Rationale: controls to be sure that each of the
secondary antibodies-fluorophore conjugates
reacts only with its corresponding primary
antibody and not with the non-corresponding
secondary antibody.
 Incubate sections as follows:
 • mouse anti-p75NTR followed by the cocktail of Donkey
anti-mouse IgG conjugated to Cy3 and Donkey anti-
rabbit IgG conjugated to 488;
 • rabbit anti-S100 followed by the cocktail of Donkey
anti-mouse IgG conjugated to Cy3 and Donkey anti-
rabbit IgG conjugated to 488.
 The expectation is labeled cells in the appropriate
channel and blank sections in the non-
corresponding channel (e.g., if the primary
antiserum has been mouse anti-p75NTR,
fluorescing cells are expected in the Cy3 channel
while the Cy2 (488) channel should remain
devoid of labeled cells). If positively stained cells
 D. Control to determine
whether interaction occurs
of fluorophores
 Rationale: Interactions like competing or
quenching fluorophores cannot be ruled
out unless a proper control experiment is
conducted. Incubate sections as follows:
 • cocktail of mouse anti-P75NTR and rabbit
anti-P75NTR followed by incubation with a
cocktail of donkey anti-mouse IgG conjugated
to Cy3 and donkey anti-rabbit IgG conjugated
to 488;
 • only with mouse anti-P75NTR followed by
incubation with a cocktail of donkey anti-
mouse IgG conjugated to Cy3 and donkey anti-
mouse IgG conjugated to 488.
 Expected results: 100% co-localization of
both markers.
 E. Control to determine
autofluorescence
 Sections were incubated with a
mixture of primary antibodies but
without secondary antibodies.
 Expected results: weak fluorescence
or blank.
 F. Control to monitor
tissue-IgG interactions
 Rationale: finally, a secondary antibody
could directly bind to tissue epitopes
rather than to its corresponding primary
antibody. To suppress this type of
interaction, the sections were pre-
incubated with excess of normal donkey
serum. In addition control experiments
should be performed.
 Treat sections as follows:
 • incubate with the vehicle of the primary
antibody (primary antibody omitted, only
blocking buffer) followed by incubation
with a cocktail of Donkey IgGs conjugated
to Cy3 and 488.
 Methods developed to double
labeling
1. Adjacent thin sections are separately stained and
1. Adjacent thin sections are separately stained and
compared (serial sectioning and mirror
sectioning methods )
2. primary antibodies are monoclonal antibodies
belonging to different immunoglobulin
subclasses
3. primary antibodies are directly labeled with
enzymes, fluorophores, or haptens such as biotin
4. primary and secondary antibodies are
sequentially applied and stained (sequential
staining method )
5. highly sensitive tyramide signal amplification is
used to distinguish primary antibodies
6. soluble immune complexes consisting of primary
and secondary monovalent Fab fragment
antibodies
7. Combination with #3 and#2 if using one primary
Triple immunofluorescence
method
 Three kinds of antibodies from different host: for
example: with one mouse (rat) monoclonal
antibody, one goat (sheep, chicken) polyclonal
antibody, and another rabbit polyclonal antibody
 With two mouse monoclonal antibodies (different
isotypes, IgG1-IgG2a-IgG2b-IgM) and another
rabbit polyclonal antibody
 based on differences between both primary antibodies:
animal species, mouse Ig isotype or IgG subclasses,
conjugates.
 With one mouse monoclonal antibodies and
another two rabbit polyclonal antibodies
 With two mouse monoclonal antibodies (same
subclass) and another rabbit polyclonal antibody
 With three monoclonal antibodies (same
subclass) or rabbit polyclonal antibodies
 Successful Strategies for
double/triple labeling with
primary antibodies from
identical species
 Three antigens in a single tissue
preparation, using unconjugated primary
antibodies raised in the same species
(Brouns et al., 2002) or raised in two
species (Nakamura and Uchihara, 2004) or
detecting two antigens by antibodies from
same species (Toth and Mezey, 2007) by
super-sensitive IHC
 double-label fluorescent IHC with identical
species-derived primary antibodies by
soluble immune complex method (Brown
 References
 Brouns, I., Van Nassauw, L., Van Genechten, J., Majewski,
M., Scheuermann, D. W., Timmermans, J. P. and
Adriaensen, D. (2002). Triple immunofluorescence staining with
antibodies raised in the same species to study the complex
innervation pattern of intrapulmonary chemoreceptors. J
Histochem Cytochem 50, 575-82.
 Brown, J. K., Pemberton, A. D., Wright, S. H. and Miller, H.
R. P. (2004). Primary Antibody-Fab Fragment Complexes: A
Flexible Alternative to Traditional Direct and Indirect
Immunolabeling Techniques. J. Histochem. Cytochem. 52, 1219-
1230.
 Ino, H. (2004). Application of Antigen Retrieval by Heating for
Double-label Fluorescent Immunohistochemistry with Identical
Species-derived Primary Antibodies. J. Histochem. Cytochem. 52,
1209-1217.
 Nakamura, A. and Uchihara, T. (2004). Dual enhancement of
triple immunofluorescence using two antibodies from the same
species. J Neurosci Methods 135, 67-70.
 Toth, Z. E. and Mezey, E. (2007). Simultaneous visualization of
multiple antigens with tyramide signal amplification using
antibodies from the same species. J Histochem Cytochem 55, 545-
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