Multiple-labeling witMultiple-labeling with Immunofluorescenh Immunofluorescence: ce: Methodological ConsiMethodological Consi

derationsderationsHenry LiHenry Li

2008-03-312008-03-31

Introduction to ImmunohistochemistrIntroduction to Immunohistochemistry y

http://www.ihcworld.com/introductiohttp://www.ihcworld.com/introduction.htmn.htm Immunohistochemistry is the localization of antigens Immunohistochemistry is the localization of antigens

or proteins in tissue sections by the use of labeled antor proteins in tissue sections by the use of labeled antibodies as specific reagents through antigen-antibody ibodies as specific reagents through antigen-antibody interactions that are visualized by a marker such as flinteractions that are visualized by a marker such as fluorescent dye, enzyme, or colloidal gold. uorescent dye, enzyme, or colloidal gold.

IntroductionIntroductionTissueTissue Preparation Preparation (Fixation, Sectioning,(Fixation, Sectioning, Whole Mount Preparation) Whole Mount Preparation)

Antigen Antigen RetrievalRetrievalIHC Methods IHC Methods (Blocking, Controls, D(Blocking, Controls, Direct Method, Indirect Method, PAP Method, ABC Meirect Method, Indirect Method, PAP Method, ABC Method, LSAB Method, Polymeric Method, CSA Method, thod, LSAB Method, Polymeric Method, CSA Method, Sensitivity Chart)Sensitivity Chart)

Multiple LabelingMultiple Labeling Immuno-Electron MicroscopyImmuno-Electron Microscopy

Tissue Preparation:- Tissue Preparation:- FixationFixation Tissue preparation is the Tissue preparation is the cornerstonecornerstone of immunohistochemistry. To ensure t of immunohistochemistry. To ensure t

he preservation of tissue architecture and cell morphology, prompt and adequate he preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significafixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability. ntly diminish the antibody binding capability.

There is There is no one universal fixativeno one universal fixative that is ideal for the demonstration of al that is ideal for the demonstration of all antigens. However, in general, many antigens can be successfully demonstratel antigens. However, in general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. The discover and developd in formalin-fixed paraffin-embedded tissue sections. The discover and development of antigen retrieval techniques further enhanced the use of formalin as roument of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories. tine fixative for immunohistochemistry in many research laboratories.

For best results, vertebrate tissues (especially neuronal tissues) usually require fiFor best results, vertebrate tissues (especially neuronal tissues) usually require fixation by transcardial perfusion for xation by transcardial perfusion for optimal tissue preservationoptimal tissue preservation. The most co. The most common fixatives used for immunohistochemistry are the followings:mmon fixatives used for immunohistochemistry are the followings:

a) 4% paraformaldehyde in 0.1M phosphate buffera) 4% paraformaldehyde in 0.1M phosphate buffer b) 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate bufferb) 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 phc) PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M ph

osphate bufferosphate buffer d) 4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistrd) 4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistr

y)y) Some antigens will not survive even moderate amounts of aldehyde fixation. UndSome antigens will not survive even moderate amounts of aldehyde fixation. Und

er this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut er this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose. The sections should be kept frozwith a cryostat without infiltrating with sucrose. The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol. After fixation, the en at -20 C or lower until fixation with cold acetone or alcohol. After fixation, the sections can be processed using standard immunohistochemical staining protocosections can be processed using standard immunohistochemical staining protocolsls

SectioningSectioning Since its introduction, paraffin wax has remained the most widely used embeddSince its introduction, paraffin wax has remained the most widely used embedd

ing medium for diagnostic histopathology in routine histological laboratories. Aing medium for diagnostic histopathology in routine histological laboratories. Accordingly, the largest proportion of material for immunohistochemistry is forccordingly, the largest proportion of material for immunohistochemistry is formalin-fixed, paraffin-embedded. malin-fixed, paraffin-embedded. Paraffin sectionsParaffin sections produce satisfactory resu produce satisfactory results for the demonstration of majority of tissue antigens with the use of antigen rlts for the demonstration of majority of tissue antigens with the use of antigen retrieval techniques.etrieval techniques.

Certain cell antigens do not survive routine fixation and paraffin embedding. So Certain cell antigens do not survive routine fixation and paraffin embedding. So the use of the use of frozen sectionsfrozen sections still remains essential for the still remains essential for the demonstration of mdemonstration of many antigensany antigens. However, the disadvantage of frozen sections includes . However, the disadvantage of frozen sections includes poor mopoor morphology, poor resolution at higher magnifications, special storrphology, poor resolution at higher magnifications, special storage needed, limited retrospective studies and cutting difficulty age needed, limited retrospective studies and cutting difficulty over paraffin sectionsover paraffin sections. .

Vibratome sectionsVibratome sections have some advantages when doing immunohistochemi have some advantages when doing immunohistochemistry since the tissue is not processed through organic solvents or high heat, whicstry 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 h can destroy the antigenicity. In addition, the morphology of tissue sections is not disrupted due to no freezing and thawing needed. Vibratome sections are ofnot disrupted due to no freezing and thawing needed. Vibratome sections are often used for floating immunostaining, especially for pre-embedding EM immunten used for floating immunostaining, especially for pre-embedding EM immunohistochemistry. The disadvantage of vibratome sections is that the sectioning pohistochemistry. The disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues. In addiction, the crocess is slow and difficult with soft and poorly fixed tissues. In addiction, the chatter marks or vibratome lines are often appeared in the sections.hatter marks or vibratome lines are often appeared in the sections.

Whole Mount PreparWhole Mount Preparationation

Small blocks of tissue (less than 5 mm thick) can bSmall blocks of tissue (less than 5 mm thick) can be processed as whole mounts. The advantage of e processed as whole mounts. The advantage of whole mount preparations is that the results prowhole mount preparations is that the results provide three dimensional information about the lovide three dimensional information about the location of antigens without the need for reconstrcation of antigens without the need for reconstruction from sections. However, the major limitatuction from sections. However, the major limitation of using whole mounts is ion of using whole mounts is antibody penetrantibody penetration may not be complete in the tissueation may not be complete in the tissue, re, resulting in uneven staining or false negative stainsulting in uneven staining or false negative staining. So Triton X-100 or saponin treatment are using. So Triton X-100 or saponin treatment are used routinely for whole mount immunohistocheed routinely for whole mount immunohistochemistry to enhance penetration of the antibody.mistry to enhance penetration of the antibody.

Antigen RetrievalAntigen Retrieval

The demonstration of many antigens can be significThe demonstration of many antigens can be significantly improved by the pretreatment with the antigeantly improved by the pretreatment with the antigen retrieval reagent that break the protein cross-linkn retrieval reagent that break the protein cross-links formed by formalin fixation and thereby uncover s formed by formalin fixation and thereby uncover hidden antigenic sites. The techniques involved the hidden antigenic sites. The techniques involved the application of heat for varying lengths of time to forapplication of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in amalin-fixed, paraffin-embedded tissue sections in an aqueous solution (commonly referred to as the retn aqueous solution (commonly referred to as the retrieval solution). This is called "rieval solution). This is called "Heat Induced EpitopHeat Induced Epitope Retrievale Retrieval (HIER)". Another method uses enzyme d (HIER)". Another method uses enzyme digestion and is called "igestion and is called "Proteolytic Induced Epitope RProteolytic Induced Epitope Retrievaletrieval (PIER)". (PIER)".

BlockingBlocking

Choice: normal host (Choice: normal host ( species of thespecies of the 22ndnd antibody) serum with BSA and oth antibody) serum with BSA and other supplement er supplement

Caution: Caution: Never block with normal serNever block with normal serum or IgG from the host species of the um or IgG from the host species of the primary antibody when using a labeleprimary antibody when using a labeled, secondary antibody. d, secondary antibody.

Basic Antibody StructureBasic Antibody Structure

How to Choose the Best Secondary Antibody How to Choose the Best Secondary Antibody for for

Immunohistochemistry ApplicationImmunohistochemistry Application Selection of the best secondary antibody can improve immunostaining and reduce false pSelection of the best secondary antibody can improve immunostaining and reduce false p

ositive or negative staining. Here is some useful information that will help you to choose ositive or negative staining. Here is some useful information that will help you to choose the best secondary antibody possible for your specific immunohistochemical application.the best secondary antibody possible for your specific immunohistochemical application. http://www.ihcworld.com/_technical_tips/2nd_antibody_tips.htm http://www.ihcworld.com/_technical_tips/2nd_antibody_tips.htm

Species of Primary Antibody: Species of Primary Antibody: The secondary antibody should be against the species that primary antibody is raised. FoThe secondary antibody should be against the species that primary antibody is raised. Fo

r example, if the primary antibody is raised in mouse, an anti-mouse secondary antibody r 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.should be used. If it is raised in rabbit, an anti-rabbit secondary antibody should be used.

Class and Subclass of Primary Antibody: Class and Subclass of Primary Antibody: The secondary antibody should match the class or subclass of the primary antibody used.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 i 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. s 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, IgIf 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 tG2b, 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 recognizhe primary antibody are not known, the anti-mouse IgG may be used since they recognize most of mouse IgG subtypes.e most of mouse IgG subtypes.

One can also use a secondary antibody specific to the IgG subclass of the primary antibodOne can also use a secondary antibody specific to the IgG subclass of the primary antibod

y. For example, if the primary antibody is mouse IgG2a, an anti-mouse IgG2a secondary ay. 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.ntibody 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 immPolyclonal antibodies (such as rabbit, goat, sheep or donkey) are typically IgG class imm

unoglobulins so the anti-IgG secondary antibodies for these species may be used.unoglobulins so the anti-IgG secondary antibodies for these species may be used.

Adsorbed Secondary AntibodAdsorbed Secondary Antibody:y:

Some of the secondary antibodies have been adsorbed with anSome of the secondary antibodies have been adsorbed with animal or human IgG. These antibodies are designed for particulimal or human IgG. These antibodies are designed for particular applications to reduce non-specific background staining. Foar applications to reduce non-specific background staining. For example, if working with rat tissues or cells, choose a secondr example, if working with rat tissues or cells, choose a secondary antibody that has been adsorbed with rat serum or IgG. Hoary antibody that has been adsorbed with rat serum or IgG. However, such adsorbed antibodies have greatly reduced epitope wever, such adsorbed antibodies have greatly reduced epitope recognition and may recognize some subclasses of IgG very wrecognition and may recognize some subclasses of IgG very weakly, especially those subclasses which are most closely homeakly, especially those subclasses which are most closely homologous to the species they were adsorbed against. For examplologous to the species they were adsorbed against. For example, do not use an anti-mouse IgG that has been adsorbed againse, do not use an anti-mouse IgG that has been adsorbed against rat IgG unless you are trying to detect a mouse primary antibt rat IgG unless you are trying to detect a mouse primary antibody in rat tissue that contains rat immunoglobulin, or in some ody in rat tissue that contains rat immunoglobulin, or in some other tissue in the presence of a rat primary antibody. Converother tissue in the presence of a rat primary antibody. Conversely, if you wish to detect a mouse primary antibody in the abssely, if you wish to detect a mouse primary antibody in the absence of rat immunoglobulins, it is best to use an anti-mouse seence of rat immunoglobulins, it is best to use an anti-mouse secondary antibody that has not been adsorbed against rat.condary antibody that has not been adsorbed against rat.

Whole IgG or F(ab')2 FragmentWhole IgG or F(ab')2 Fragments of the Secondary Antibody s of the Secondary Antibody

Anti-IgG (H+L): Anti-IgG (H+L): This antibody reacts with both the heavy and This antibody reacts with both the heavy and light chains of the IgG molecule, i.e. it reacts with both the Fc 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 othand F(ab')2 portions of IgG. Anti-IgG (H+L) also reacts with other immunoglobulin classes (e.g. IgM, IgA, etc.) since all immuer immunoglobulin classes (e.g. IgM, IgA, etc.) since all immunoglobulins share the same light chains (either kappa or lambnoglobulins share the same light chains (either kappa or lambda). This is commonly used secondary antibody.da). This is commonly used secondary antibody.

Anti-IgG, Fc fragment specific:Anti-IgG, Fc fragment specific: This antibody reacts with the This antibody reacts with the Fc portion of the IgG heavy chain. In each case, it has been adFc portion of the IgG heavy chain. In each case, it has been adsorbed against F(ab')2 fragments. In some cases, the antibodiesorbed against F(ab')2 fragments. In some cases, the antibodies are additionally adsorbed to minimize possible cross-reactivis are additionally adsorbed to minimize possible cross-reactivity to IgM and IgA, or to IgM alone.ty to IgM and IgA, or to IgM alone.

Anti-IgG, F(ab')2 fragment specific: Anti-IgG, F(ab')2 fragment specific: This antibody has been aThis antibody has been adsorbed against Fc fragments and therefore reacts only with tdsorbed against Fc fragments and therefore reacts only with the Fab portion of IgG. Since it reacts with light chains, it also rhe Fab portion of IgG. Since it reacts with light chains, it also reacts with other immunoglobulins sharing the same light chaieacts with other immunoglobulins sharing the same light chains. This antibody is used for specific applications such as douns. This antibody is used for specific applications such as double labeling methods or for tissues or cells that have Fc receptble labeling methods or for tissues or cells that have Fc receptors (thymus, spleen). ors (thymus, spleen).

How to Choose a Secondary AnHow to Choose a Secondary Antibody tibody

1.1. In what animal was the primary antibody developed?In what animal was the primary antibody developed?

2.2. What is the class and/or subclass of the primary antibodyWhat is the class and/or subclass of the primary antibody :This is primarily important :This is primarily important in the case of monoclonal antibodies. Polyclonal antibodies (generally developed in rabbin the case of monoclonal antibodies. Polyclonal antibodies (generally developed in rabbit, goat, sheep or donkey) are typically IgG class immunoglobulins. For this reason, the sit, 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 econdary 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 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 thIgG 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 e 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) secondaand/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.ry antibodies may be used since they recognize most mouse immunoglobulin subtypes.

3.3. What form of antibody should be used?What form of antibody should be used?

4.4. What kind of label?What kind of label?

5.5. Is there an appropriate adsorbed secondary antibody available?Is there an appropriate adsorbed secondary antibody available?

6.6. Is a F(ab )2 fragment product necessary?Is a F(ab )2 fragment product necessary?

7.7. If all else is equal, decide based on price/titer and availability.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.htmlhttp://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Antibody_Explorer/Technical_Support/Secondary_Antibody.html

Selection and Location of Affinity-PSelection and Location of Affinity-Purifiedurified

AntibodiesAntibodieshttp://www.jacksonimmuno.com/technical/select.http://www.jacksonimmuno.com/technical/select.aspasp

Step 1 Select either "Whole Molecules" or "F(aStep 1 Select either "Whole Molecules" or "F(ab')2 Fragments" of the antibodyb')2 Fragments" of the antibody

Step 2 Find the species of the antigen with whStep 2 Find the species of the antigen with which you would like the antibody to reactich you would like the antibody to react

Step 3 Select the host species in which you waStep 3 Select the host species in which you want the antibody to be madent the antibody to be made

Step 4 Select most appropriate antibody from Step 4 Select most appropriate antibody from the multiple choices:the multiple choices: to minimize cross-reactito minimize cross-reactivityvity

Step 5Step 5 Find the price, size, and code number Find the price, size, and code number of the selected antibodyof the selected antibody

ControlsControls Immunocytochemistry is used for antibody localization Immunocytochemistry is used for antibody localization

of proteins in cells and tissues. of proteins in cells and tissues. The specificity of the resThe specificity of the results depends on two independent criteriaults depends on two independent criteria: : the specificity the specificity of the antibody and of the method usedof the antibody and of the method used. The antibody s. The antibody specificity is best determined by immunoblot and or impecificity is best determined by immunoblot and or immunoprecipitation. Absorption of the antibody with a pmunoprecipitation. Absorption of the antibody with a protein does not determine that the antibody would have rotein does not determine that the antibody would have bound to the same protein in the tissue, and therefore is bound to the same protein in the tissue, and therefore is not a good control for antibody specificity. not a good control for antibody specificity. The specificiThe specificity of the methodty of the method is best determined by both a is best determined by both a negative cnegative controlontrol, replacing the primary antibody with serum, and , replacing the primary antibody with serum, and a a positive controlpositive control, using the antibody with cells known t, using the antibody with cells known to contain the protein. With the increasing use of immuno contain the protein. With the increasing use of immunocytochemistry, it is important to be aware of the approocytochemistry, it is important to be aware of the appropriate controls needed to show specificity of the labelinpriate controls needed to show specificity of the labeling (Burry 2000).g (Burry 2000).

Burry RW. 2000. Specificity Controls for ImmunocytochBurry RW. 2000. Specificity Controls for Immunocytochemical Methods. J Histochem Cytochem 48(2):163-166.emical Methods. J Histochem Cytochem 48(2):163-166.

ControlsControls ***Special controls***Special controls must be run in order to test the protocol and for th must be run in order to test the protocol and for th

e specificity of the antibody being used.e specificity of the antibody being used. Positive controlPositive control is to test a protocol or procedure and make sure it works. It 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 cwill 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 bontrol tissue showed negative staining, the protocol or procedure needs to be checked until a good positive staining is obtained.e checked until a good positive staining is obtained.

Negative controlNegative control is to test for the specificity of an antibody involved. First, n is to test for the specificity of an antibody involved. First, n

o staining must be shown when omitting primary antibody or replacing an so staining must be shown when omitting primary antibody or replacing an specific primary antibody with normal serum (must be the same species as ppecific primary antibody with normal serum (must be the same species as primary antibody). This control is easy to achieve and can be used routinely irimary antibody). This control is easy to achieve and can be used routinely in immunohistochemical staining.n immunohistochemical staining.

Second, the staining must be inhibited by adsorption of a primary antibody 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 rewith 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 necesslated or unrelated antigens. This type of negative control is ideal and necessary in the characterization and evaluation of new antibodies but it is sometiary in the characterization and evaluation of new antibodies but it is sometimes difficult to obtain the purified antigen, therefore it is rarely used routinmes difficult to obtain the purified antigen, therefore it is rarely used routinely in immunohistochemical staining. ely in immunohistochemical staining. Adsorption controls as minimally accAdsorption controls as minimally acceptable documentation,eptable documentation, however, the power of adsorption test is subject to a however, the power of adsorption test is subject to arguing in the present .rguing in the present .

Control for AntibodiesControl for Antibodies WHAT IS AN ADEQUATE SET OF CONTROLS FWHAT IS AN ADEQUATE SET OF CONTROLS F

OR AN ANTIBODY? OR AN ANTIBODY? Does the antibody actually reveal the antigDoes the antibody actually reveal the antigen of interest, or is the binding an artifact? The gold standard for decidinen 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. g this issue is to stain tissue with and without the antigen of interest.

Repeat the experiment for the purpose of verifRepeat the experiment for the purpose of verification ication

DON'T BELIEVE EVERYTHING YOU SEE:DON'T BELIEVE EVERYTHING YOU SEE: it is impit is important to have sufficient controls to convince a skeptical observer that the ortant to have sufficient controls to convince a skeptical observer that the staining pattern observed really does represent the antigen, and wherevestaining pattern observed really does represent the antigen, and wherever possible the results should be checked with other techniques.r possible the results should be checked with other techniques. ((unless unless

you understand how the images were captured and processed)you understand how the images were captured and processed) Clifford BS, Sawchenko PE. 2003. Magic peptides, magic Clifford BS, Sawchenko PE. 2003. Magic peptides, magic

antibodies: Guidelines for appropriate controls for imantibodies: Guidelines for appropriate controls for immunohistochemistry. J Com Neurol 465(2):161-163.munohistochemistry. J Com Neurol 465(2):161-163.

Introduction to Multiple LaIntroduction to Multiple Labeling beling It is often useful to be able to stain for two or more antigIt is often useful to be able to stain for two or more antig

ens in one common tissue section. This can be achieved ens in one common tissue section. This can be achieved by by immunofluorescence methodimmunofluorescence method using different fl using different fluorescent dyes. Multiple staining can also be done with uorescent dyes. Multiple staining can also be done with peroxidase conjugated antibodies developed with differperoxidase conjugated antibodies developed with different chromogen substrates to produce the end products ent chromogen substrates to produce the end products of different colors. There are three basic approaches in of different colors. There are three basic approaches in planning multiple staining: parallel, sequential and adjaplanning multiple staining: parallel, sequential and adjacent. In addition, the antibody dilution and conditioncent. In addition, the antibody dilution and condition arare also important factors to be considered. Finally, appre also important factors to be considered. Finally, appropriate color combination is also crucial since improper opriate color combination is also crucial since improper color combination may produce poor result and fail to dcolor combination may produce poor result and fail to demonstrate multiple antigens in the same section. For bemonstrate multiple antigens in the same section. For best result, the careful design and test of multiple staininest result, the careful design and test of multiple staining protocols are necessaryg protocols are necessary. .

Whatever labelling combination is employed, Whatever labelling combination is employed, it is essential to conduct control experiments it is essential to conduct control experiments to ensure that there is no antibody cross to ensure that there is no antibody cross reactivityreactivity

Controls for confocal laser Controls for confocal laser scanning microscope scanning microscope

(CLSM)(CLSM)Multiple immunofluorescence labeling in combination Multiple immunofluorescence labeling in combination

with CLSM is a powerful strategy to visualize the with CLSM is a powerful strategy to visualize the spatial relationship between different antigens in spatial relationship between different antigens in histological sections.histological sections.

Samples: Samples: Dorsal root ganglia from adult Sprague-Delay ratsDorsal root ganglia from adult Sprague-Delay rats

Primary Antibodies: Primary Antibodies: p75NTR monoclonal antibody (IgG192, IgG1); Rabbit anti S100 p75NTR monoclonal antibody (IgG192, IgG1); Rabbit anti S100

and and rabbit anti-P75NTR (#9991) rabbit anti-P75NTR (#9991) ployclonal antibodies.ployclonal antibodies. Secondary antibodies: Secondary antibodies:

Donkey anti-mouse IgG(H+L) conjugated to Cy3 (Jackson Donkey anti-mouse IgG(H+L) conjugated to Cy3 (Jackson Immuno)Immuno)

Donkey anti-mouse IgG(H+L) conjugated to Donkey anti-mouse IgG(H+L) conjugated to Alexa Fluor 488 Alexa Fluor 488 (probes-Invitrogen)(probes-Invitrogen)

Donkey anti-rabbit IgG(H+L) conjugated to Donkey anti-rabbit IgG(H+L) conjugated to Alexa Fluor 488 Alexa Fluor 488 (probes-Invitrogen)(probes-Invitrogen)

A. Control for specificity I—A. Control for specificity I—positive controlpositive control

Rationale:Rationale: in every control experiment a separate positive control check has 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 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 in the appropriate channel in the CLSM in the first place (a channelchannel is a is a specific setup of the CLSM including illumination with one of the lasers and 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 the filter and mirror settings specific for that particular laser and its corresponding fluorophore-to-be-detected). It is important to know a priori corresponding fluorophore-to-be-detected). It is important to know a priori that the immunocytochemical reactions have been successfully completed that the immunocytochemical reactions have been successfully completed since otherwise no firm conclusions about the degree of success of the since otherwise no firm conclusions about the degree of success of the immunocytochemical procedures can be drawn when no image is obtained in immunocytochemical procedures can be drawn when no image is obtained in the CLSM. the CLSM.

Next to determining the specificity of the primary-secondary antibody Next to determining the specificity of the primary-secondary antibody binding, these sections are used to check the specificity of the parameters of 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 the CLSM: does the right fluorophore produce a detector signal with the corresponding laser and filter settings, while it produces no detector signal corresponding laser and filter settings, while it produces no detector signal when illuminated with the non-corresponding (second) laser seen through the when illuminated with the non-corresponding (second) laser seen through the filter settings belonging to the second laser's setup.filter settings belonging to the second laser's setup.

Incubate sections as follows: Incubate sections as follows: • • mouse anti-p75NTR antibodies followed by incubation with Donkey anti-mouse anti-p75NTR antibodies followed by incubation with Donkey anti-

mouse IgG conjugated to Cy3;mouse IgG conjugated to Cy3; • • rabbit anti-S100 antibodies followed by incubation with Donkey anti-rabbit rabbit anti-S100 antibodies followed by incubation with Donkey anti-rabbit

IgG conjugated to 488IgG conjugated to 488 Expected results:Expected results: labeled cells only in the corresponding channel and no labeled cells only in the corresponding channel and no

labeling of cells in the non-corresponding channel.labeling of cells in the non-corresponding channel.

Control for specificity II-non Control for specificity II-non crossing reaction controlcrossing reaction control

Rationale:Rationale: controls to be sure that each of the controls to be sure that each of the primary antibodies does not react with the non-primary antibodies does not react with the non-corresponding secondary antibody-conjugate. corresponding secondary antibody-conjugate.

Incubate sections as follows: Incubate sections as follows: • • mouse anti-p75NTR followed by incubation with mouse anti-p75NTR followed by incubation with

donkey anti-rabbit IgG conjugated to 488;donkey anti-rabbit IgG conjugated to 488; • • rabbit anti-S100 followed by incubation with rabbit anti-S100 followed by incubation with

donkey anti-mouse IgG conjugated to cy3.donkey anti-mouse IgG conjugated to cy3. Expected results:Expected results: blank sections. If labeled cells blank sections. If labeled cells

appear in any of these sections, then the “wrong” appear in any of these sections, then the “wrong” secondary antibody binds to the primary antibody. It secondary antibody binds to the primary antibody. It should be noted that these sections must be should be noted that these sections must be observed in the CLSM at a high laser illumination observed in the CLSM at a high laser illumination setting in order to detect even traces of fluorescencesetting in order to detect even traces of fluorescence..

C. Control to monitor IgG C. Control to monitor IgG interactionsinteractions

Rationale:Rationale: controls to be sure that each of the secondary controls to be sure that each of the secondary antibodies-fluorophore conjugates reacts only with its antibodies-fluorophore conjugates reacts only with its corresponding primary antibody and not with the non-corresponding primary antibody and not with the non-corresponding secondary antibody. corresponding secondary antibody.

Incubate sections as follows: Incubate sections as follows: • • mouse anti-p75NTR followed by the cocktail of Donkey anti-mouse anti-p75NTR followed by the cocktail of Donkey anti-

mouse IgG conjugated to Cy3 and Donkey anti-rabbit IgG mouse IgG conjugated to Cy3 and Donkey anti-rabbit IgG conjugated to 488;conjugated to 488;

• • rabbit anti-S100 followed by the cocktail of Donkey anti-mouse rabbit anti-S100 followed by the cocktail of Donkey anti-mouse IgG conjugated to Cy3 and Donkey anti-rabbit IgG conjugated to IgG conjugated to Cy3 and Donkey anti-rabbit IgG conjugated to 488.488.

The expectation is labeled cells in the appropriate The expectation is labeled cells in the appropriate channel and blank sections in the non-corresponding channel and blank sections in the non-corresponding channel (e.g., if the primary antiserum has been mouse channel (e.g., if the primary antiserum has been mouse anti-p75NTR, fluorescing cells are expected in the Cy3 anti-p75NTR, fluorescing cells are expected in the Cy3 channel while the Cy2 (488) channel should remain channel while the Cy2 (488) channel should remain devoid of labeled cells). If positively stained cells are devoid of labeled cells). If positively stained cells are visible in the non-corresponding channel, this is indicative visible in the non-corresponding channel, this is indicative for binding of secondary antibodies to each other.for binding of secondary antibodies to each other.

D. Control to determine D. Control to determine whether interaction occurs whether interaction occurs

of fluorophores of fluorophores Rationale:Rationale: Interactions like competing or Interactions like competing or

quenching fluorophores cannot be ruled out quenching fluorophores cannot be ruled out unless a proper control experiment is unless a proper control experiment is conducted. Incubate sections as follows: conducted. Incubate sections as follows: • • cocktail of mouse anti-P75NTR and rabbit anti-cocktail of mouse anti-P75NTR and rabbit anti-

P75NTR followed by incubation with a cocktail P75NTR followed by incubation with a cocktail of donkey anti-mouse IgG conjugated to Cy3 and of donkey anti-mouse IgG conjugated to Cy3 and donkey anti-rabbit IgG conjugated to 488;donkey anti-rabbit IgG conjugated to 488;

• • only with mouse anti-P75NTR followed by only with mouse anti-P75NTR followed by incubation with a cocktail of donkey anti-mouse incubation with a cocktail of donkey anti-mouse IgG conjugated to Cy3 and donkey anti-mouse IgG conjugated to Cy3 and donkey anti-mouse IgG conjugated to 488.IgG conjugated to 488.

Expected results:Expected results: 100% co-localization of 100% co-localization of both markers.both markers.

E. Control to determine E. Control to determine autofluorescence autofluorescence

Sections were incubated with a Sections were incubated with a mixture of primary antibodies but mixture of primary antibodies but without secondary antibodies. without secondary antibodies.

Expected results:Expected results: weak fluorescence weak fluorescence or blank. or blank.

F. Control to monitor F. Control to monitor tissue-IgG interactionstissue-IgG interactions

Rationale:Rationale: finally, a secondary antibody could finally, a secondary antibody could directly bind to tissue epitopes rather than to directly bind to tissue epitopes rather than to its corresponding primary antibody. To its corresponding primary antibody. To suppress this type of interaction, the sections suppress this type of interaction, the sections were pre-incubated with excess of normal were pre-incubated with excess of normal donkey serum. In addition control experiments donkey serum. In addition control experiments should should be performed. be performed.

Treat sections as follows: Treat sections as follows: • • incubate with the vehicle of the primary incubate with the vehicle of the primary

antibody (primary antibody omitted, only antibody (primary antibody omitted, only blocking buffer) followed by incubation with a blocking buffer) followed by incubation with a cocktail of Donkey IgGs conjugated to Cy3 and cocktail of Donkey IgGs conjugated to Cy3 and 488.488.

Expected results: blank sections.Expected results: blank sections.

Methods developed to double Methods developed to double labelinglabeling

1.1. Adjacent thin sections are separately stained and Adjacent thin sections are separately stained and compared (serial sectioning and mirror sectioning compared (serial sectioning and mirror sectioning methods )methods )

2.2. primary antibodies are monoclonal antibodies belonging to primary antibodies are monoclonal antibodies belonging to different immunoglobulin subclasses different immunoglobulin subclasses

3.3. primary antibodies are directly labeled with enzymes, primary antibodies are directly labeled with enzymes, fluorophores, or haptens such as biotin fluorophores, or haptens such as biotin

4.4. primary and secondary antibodies are sequentially applied primary and secondary antibodies are sequentially applied and stained (sequential staining method )and stained (sequential staining method )

5.5. highly sensitive tyramide signal amplification is used to highly sensitive tyramide signal amplification is used to distinguish primary antibodies distinguish primary antibodies

6.6. soluble immune complexes consisting of primary and soluble immune complexes consisting of primary and secondary monovalent Fab fragment antibodies secondary monovalent Fab fragment antibodies

7.7. Combination with #3 and#2 if using one primary antibody Combination with #3 and#2 if using one primary antibody is conjugatedis conjugated

8.8. Combination #5 and #6 if un-conjugated primary Combination #5 and #6 if un-conjugated primary antibodies usedantibodies used

Triple immunofluorescence Triple immunofluorescence methodmethod

Three kinds of antibodies from different host: for examThree kinds of antibodies from different host: for example: with one ple: with one mouse (rat) monoclonal antibody, one mouse (rat) monoclonal antibody, one goat (sheep, chicken) polyclonal antibody, and goat (sheep, chicken) polyclonal antibody, and another rabbit polyclonal antibodyanother rabbit polyclonal antibody

With two mouse monoclonal antibodies (different With two mouse monoclonal antibodies (different isotypes, IgG1-IgG2a-IgG2b-IgM) and another rabbit isotypes, IgG1-IgG2a-IgG2b-IgM) and another rabbit polyclonal antibodypolyclonal antibody based on differences between both primary antibodies: based on differences between both primary antibodies:

animal species, mouse Ig isotype or IgG subclasses, animal species, mouse Ig isotype or IgG subclasses, conjugates.conjugates.

With one mouse monoclonal antibodies and another With one mouse monoclonal antibodies and another two rabbit polyclonal antibodiestwo rabbit polyclonal antibodies

With two mouse monoclonal antibodies (same With two mouse monoclonal antibodies (same subclass) and another rabbit polyclonal antibodysubclass) and another rabbit polyclonal antibody

With three monoclonal antibodies (same subclass) or With three monoclonal antibodies (same subclass) or rabbit polyclonal antibodiesrabbit polyclonal antibodies

Successful Strategies for Successful Strategies for double/triple labeling with double/triple labeling with primary antibodies from primary antibodies from

identical speciesidentical species TThree antigens in a single tissue preparation, hree antigens in a single tissue preparation,

using unconjugated primary antibodies raised using unconjugated primary antibodies raised in the same species (Brouns et al., 2002) or in the same species (Brouns et al., 2002) or raised in two species (Nakamura and raised in two species (Nakamura and Uchihara, 2004) or detecting two antigens by Uchihara, 2004) or detecting two antigens by antibodies from same species (Toth and antibodies from same species (Toth and Mezey, 2007) by super-sensitive IHC Mezey, 2007) by super-sensitive IHC

double-label fluorescent IHC with identical double-label fluorescent IHC with identical species-derived primary antibodies by soluble species-derived primary antibodies by soluble immune complex method (Brown et al., 2004; immune complex method (Brown et al., 2004; Ino, 2004).Ino, 2004).

ReferencesReferences Brouns, I., Van Nassauw, L., Van Genechten, J., Majewski, M., Brouns, I., Van Nassauw, L., Van Genechten, J., Majewski, M.,

Scheuermann, D. W., Timmermans, J. P. and Adriaensen, D. Scheuermann, D. W., Timmermans, J. P. and Adriaensen, D. (2002). (2002). Triple immunofluorescence staining with antibodies raised Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors. intrapulmonary chemoreceptors. J Histochem CytochemJ Histochem Cytochem 50, 575-82. 50, 575-82.

Brown, J. K., Pemberton, A. D., Wright, S. H. and Miller, H. R. Brown, J. K., Pemberton, A. D., Wright, S. H. and Miller, H. R. P.P. (2004). Primary Antibody-Fab Fragment Complexes: A Flexible (2004). Primary Antibody-Fab Fragment Complexes: A Flexible Alternative to Traditional Direct and Indirect Immunolabeling Alternative to Traditional Direct and Indirect Immunolabeling Techniques. Techniques. J. Histochem. Cytochem.J. Histochem. Cytochem. 5252, 1219-1230. , 1219-1230.

Ino, H.Ino, H. (2004). Application of Antigen Retrieval by Heating for (2004). Application of Antigen Retrieval by Heating for Double-label Fluorescent Immunohistochemistry with Identical Double-label Fluorescent Immunohistochemistry with Identical Species-derived Primary Antibodies. Species-derived Primary Antibodies. J. Histochem. Cytochem.J. Histochem. Cytochem. 5252, , 1209-1217.1209-1217.

Nakamura, A. and Uchihara, T.Nakamura, A. and Uchihara, T. (2004). Dual enhancement of (2004). Dual enhancement of triple immunofluorescence using two antibodies from the same triple immunofluorescence using two antibodies from the same species. species. J Neurosci MethodsJ Neurosci Methods 135135, 67-70. , 67-70.

Toth, Z. E. and Mezey, E.Toth, Z. E. and Mezey, E. (2007). Simultaneous visualization of (2007). Simultaneous visualization of multiple antigens with tyramide signal amplification using multiple antigens with tyramide signal amplification using antibodies from the same species. antibodies from the same species. J Histochem CytochemJ Histochem Cytochem 5555, 545-, 545-54 54