Multiple-labeling With Immunofluorescence
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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 Tissue preparation is the cornerstone of the preservation of tissue 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 paraffinembedded 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, paraffinembedded 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 AffinityPurified 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:
Primary Antibodies:
Dorsal root ganglia from adult Sprague-Delay rats
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 noncorresponding 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 antirabbit IgG conjugated to 488; • rabbit anti-S100 followed by the cocktail of Donkey anti-mouse IgG conjugated to Cy3 and Donkey antirabbit IgG conjugated to 488.
The expectation is labeled cells in the appropriate channel and blank sections in the noncorresponding 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 antimouse IgG conjugated to Cy3 and donkey antimouse 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 preincubated 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.
2.
3. 4.
5. 6.
7.
Adjacent thin sections are separately stained and compared (serial sectioning and mirror sectioning methods ) primary antibodies are monoclonal antibodies belonging to different immunoglobulin subclasses primary antibodies are directly labeled with enzymes, fluorophores, or haptens such as biotin primary and secondary antibodies are sequentially applied and stained (sequential staining method ) highly sensitive tyramide signal amplification is used to distinguish primary antibodies soluble immune complexes consisting of primary and secondary monovalent Fab fragment antibodies 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, 12191230. 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, 54554
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 Tissue preparation is the cornerstone of the preservation of tissue 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 paraffinembedded 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, paraffinembedded 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 AffinityPurified 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:
Primary Antibodies:
Dorsal root ganglia from adult Sprague-Delay rats
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 noncorresponding 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 antirabbit IgG conjugated to 488; • rabbit anti-S100 followed by the cocktail of Donkey anti-mouse IgG conjugated to Cy3 and Donkey antirabbit IgG conjugated to 488.
The expectation is labeled cells in the appropriate channel and blank sections in the noncorresponding 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 antimouse IgG conjugated to Cy3 and donkey antimouse 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 preincubated 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.
2.
3. 4.
5. 6.
7.
Adjacent thin sections are separately stained and compared (serial sectioning and mirror sectioning methods ) primary antibodies are monoclonal antibodies belonging to different immunoglobulin subclasses primary antibodies are directly labeled with enzymes, fluorophores, or haptens such as biotin primary and secondary antibodies are sequentially applied and stained (sequential staining method ) highly sensitive tyramide signal amplification is used to distinguish primary antibodies soluble immune complexes consisting of primary and secondary monovalent Fab fragment antibodies 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, 12191230. 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, 54554
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