While using the right antibody to target the antigen and amplify the signal is obviously the most crucial component in reliable immunohistochemical (IHC) visualization, all of the steps in the IHC process are important for generating great images. Sub-optimal IHC staining is common, but can be corrected by adjusting some of the variables in the workflow steps, such as sample preparation, antigen retrieval and incubation times, to name a few.
Whether you are new to IHC or an experienced researcher wanting to confirm your method, consider these five critical steps to help ensure that your images are publication ready the first time.
Date: May 2, 2018
Time: 8:00am PDT
Immunohistochemistry protocols, which utilize antibodies to visualize proteins in tissue sections, have many steps that need optimization to prevent nonspecific background effects, artifacts, or inadequate detection by dyes.
How the tissue sample is prepared determines its structure and the ways in which antigens may be detected by antibodies. It’s important to choose a fixation method that is compatible with the type of tissue being used your desired experimental outcomes. Use frozen tissue or acetone fixation if you want good antigen unmasking but do not necessarily need to preserve cell morphology. Formalin-fixed and paraffin-embedded (FFPE) tissue samples are a great choice when preserving cell morphology is important in your study.
Superfrost Excell Microscope Slides
Polysine Microscope Adhesion Slides
| Possible reasons | Tips |
|---|---|
| Slides are too old | Use fresh cut slides; if they need to be stored, do so at 4°C |
| Tissue on slides dried out | Rehydrate and keep the tissue covered in buffer during staining |
| Uneven background staining might signal improper deparaffinization | Use fresh xylene and deparaffinize sections longer |
| Possible reasons | Tips |
|---|---|
| Insufficient washing; can lead to high background due to traces of fixative | Ensure at least 3 washes with PBS between steps |
| Inadequate fixation; can lead to diffusion of antigen in the tissue | Increase the time of postfixation |
| Diffuse staining; caused by tissue damage | Carefully prepare tissue sample to avoid tissue damage |
| Insufficient penetration of detection reagents | Prepare thinner sections |
| Different fixatives affect tissue antigen differently | Optimize the pH, incubation time and temperature |
Antigen unmasking is a necessary step prior to antibody labeling, as the tissue fixation process typically causes cross-linking of proteins. This is more common in formalin fixation due to the nature of the chemistry, but can easily be reversed through heat (the most commonly used method), simple buffer treatment or protease digestion. Treatment choice depends on the way the epitope configuration needed for antibody detection. This step re-exposes the epitopes on the antigen and allows antibody binding.
Lab Vision PT Module Deparaffinization and Heat-Induced Epitope Retrieval Solutions (100X)
Lab Vision Protease XXV for Enzyme-Induced Epitope Retrieval
Lab Vision Pepsin Solution for Enzyme-Induced Epitope Retrieval
eBioscience IHC Antigen Retrieval Solution - High pH (10X)
eBioscience IHC Antigen Retrieval Solution - Low pH (10X)
| Possible reasons | Tips |
|---|---|
Antigen is still masked due to protein crosslinking caused during fixation step |
Use proper antigen unmasking method, preferably via microwave; a pressure cooker can also be used, but the use of a water bath is not recommended |
Blocking endogenous enzymes and antibodies within the tissue is a critical for minimizing background staining and reducing false positive staining. This is typically achieved by incubating samples with a buffer that blocks the non-specific sites to which the primary or secondary antibodies may otherwise bind.
eBioscience IHC /ICC Blocking Buffer - Low Protein
eBioscience IHC/ICC Blocking Buffer - High Protein
Avidin/Biotin Blocking Kit
CAS-Block Histochemical Reagent
Blocker FL Fluorescent Blocking Buffer
| Possible reasons | Tips |
|---|---|
| Interference from endogenous peroxidases or phosphatases | Quench with 3% H2O2 in methanol or water or use a kit (i.e., Thermo Scientific Peroxidase Suppressor) prior to using antibodies; use enzyme inhibitors for alkaline phosphatases |
| Interference from endogenous biotin activity | Block using the avidin/biotin blocking reagent before incubating the primary antibodies |
| Interference from endogenous enzymes | Drain the excess buffer instead of washing the tissue sample prior to the addition of antibodies |
When selecting a primary antibody, ensure it has been tested in immunohistochemistry applications. The Invitrogen antibody portfolio comprises over 36,000 high-quality antibodies optimized and tested for IHC. Primary antibodies are available unconjugated (for indirect methods) or in conjugated format for direct detection methods or for multiplexing. Secondary antibodies are also available in a range of conjugates for colorimetric/chromogenic or fluorescence-based IHC.
In addition to antigen detection, cellular processes can also be analyzed in tissue sections prepared for immunohistochemistry. The use of Click chemistry, which combines EdU labeling with colorimetric detection, allows direct detection of cell proliferation in fixed tissues with conventional colorimetric stains. Additionally, the use of Click chemistry with TUNEL labeling allows colorimetric detection of fragmented DNA that occurs during apoptotic events.
Click-iT EdU Colorimetric IHC Detection Kit
Click-iT TUNEL Colorimetric IHC Detection Kit
| Possible reasons | Tips |
|---|---|
| Primary and secondary antibodies have lost their activity | Check antibody for storage conditions, contamination, change in pH or multiple freeze/thaw cycles |
| Primary antibody doesn’t work in IHC application | Make sure antibodies are listed as suitable for use in IHC application |
| Not enough antibody to label available target protein in tissue | Use adequate antibody dilution buffer and proper titration according to the data sheet recommendations |
| Insufficient primary antibody binding | Increase antibody concentration or increase incubation period to overnight at 4°C |
| Primary antibody has not been validated for all types of tissue preparation (fixation method) | Confirm IHC functional application is listed for the primary antibody and the type of IHC you plan to perform (formalin/PFA, fresh, frozen, etc.) |
| Improper species/isotype for protein target or primary antibody (if using a secondary) | Confirm compatibility of primary and secondary antibodies (species and isotype compatibility) |
| Loss of fluorescence signal | If performing fluorescent IHC, ensure the secondary Ab is stored in the dark to prevent degeneration of signal |
| The protein of interest might not be present in the tissue | Check for protein localization and run a positive control |
| The protein of interest is not abundant in the tissue | Amplify the signal using a biotin-conjugated secondary antibody and conjugated streptavidin |
| Phosphorylated protein, requires specialized phosphorylation antibody incubation conditions | Confirm posttranslational modification of target protein and check antibody-specific experimental conditions |
| The epitope might have been modified by the antigen retrieval method used and the antibody does not recognize it | Use a different antigen retrieval method (heat and pH6 or pH9 buffer, enzymatic method, etc.) |
| Protein of interest is a nuclear protein, and is unavailable to be detected by the antibody | Use Triton-X in the blocking buffer and the antibody dilution buffer to permeabilize the membranes |
| High concentration of secondary antibody can reduce antigen detection | Troubleshoot by titering concentration to check signal strength |
| Standard secondary antibodies directly conjugated to HRP may not provide enough signal amplification | Use polymer-based detection reagents or avidin/biotin–based detection systems |
| PBS buffer contains bacteria (contaminated), which can damage phospho sites on proteins | Make certain all buffers are sterile/clean and fresh; visually inspect for clarity |
| Enzyme-substrate reaction is impaired | Do not use buffers with sodium azide in the presence of HRP, and be aware that DI water can contain peroxidase inhibitors that affect enzyme activity |
| Incorrect/non-specific binding | Increase incubation period and/or change blocking agent |
| Possible reasons | Tips |
|---|---|
| Non-specific binding of primary antibodies; if the concentration is too high it elicits high-background and non-specific binding | Use higher dilution of primary antibodies and titrate to optimize concentration |
| Non-specific binding of secondary antibodies | Reduce by treating the tissue with normal serum from the same species as the secondary antibody or use Invitrogen pre-adsorbed, affinity purified secondary antibodies |
| Using antibodies on same species (i.e., mouse antibodies on mouse tissues) | Treat the tissue with mouse on mouse blocking reagent before incubating with primary antibody |
| High incubation temperatures | Decrease temperature or use 4°C incubation In fluorescent IHC |
| Formalin or paraformaldehyde fixatives cause autofluorescence in the green spectrum in fluorescent IHC | Use a fluorophore in the red spectrum or the infrared range (if IR detection system is available) |
| Non-specific binding of polyclonal antibody | Use a monoclonal primary antibody instead of a polyclonal to reduce cross-reactivity |
| Concentration of antibody is too high, causing non-specific binding | Decrease the concentrations of the primary and/or secondary antibodies to reduce nonspecific binding |
| Substrate concentration too high | Reduce the substrate incubation time and dilute the substrate further; choose a different enzyme/substrate pair if your first choice doesn’t work |
Thermo Fisher Scientific offers some of the most innovative microscopy systems for obtaining images of tissues stained by colorimetric or fluorescent labeled antibodies.
| Possible reasons | Tips |
|---|---|
| Light source/path is not set properly | Activate light source or adjust light path |
| Focus not set properly | Adjust focus |
| Filters not properly adjusted | Contact tech support for assistance with EVOS FL Auto 2 Imaging System |
| Staining did not work | Be sure to run a positive control to confirm staining method worked and check method carefully for missing or incorrectly executed steps; contact tech support for further assistance |
| Possible reasons | Tips |
|---|---|
| Objective lens not clean | Clean objective lens properly before visualization |
| Optical aberration of image | Check thickness of cover slips |
| Emission spectrum overlap in fluorescent IHC samples | Choose filter sets appropriate for the label used and choose fluorophores from separate areas of spectrum |
| Incompatibility between chromogen and mounting media leads to signal erosion | Check compatibility between substrate and mounting media; use aqueous mounting media with enzymatic and fluorescent labels or organic mounting media with enzymatic labels only; use DAB substrate with non-aqueous mounting medium and use aqueous mounting media in multiplex analysis |
| Incompatibility of mounting media and coverslip refractive index | Match the refractive index (RI) of the mounting media and the glass (RI= 1.52) as much as possible for tissue transparency and image quality; different types of mounting media have different RIs |
How the tissue sample is prepared determines its structure and the ways in which antigens may be detected by antibodies. It’s important to choose a fixation method that is compatible with the type of tissue being used your desired experimental outcomes. Use frozen tissue or acetone fixation if you want good antigen unmasking but do not necessarily need to preserve cell morphology. Formalin-fixed and paraffin-embedded (FFPE) tissue samples are a great choice when preserving cell morphology is important in your study.
Superfrost Excell Microscope Slides
Polysine Microscope Adhesion Slides
| Possible reasons | Tips |
|---|---|
| Slides are too old | Use fresh cut slides; if they need to be stored, do so at 4°C |
| Tissue on slides dried out | Rehydrate and keep the tissue covered in buffer during staining |
| Uneven background staining might signal improper deparaffinization | Use fresh xylene and deparaffinize sections longer |
| Possible reasons | Tips |
|---|---|
| Insufficient washing; can lead to high background due to traces of fixative | Ensure at least 3 washes with PBS between steps |
| Inadequate fixation; can lead to diffusion of antigen in the tissue | Increase the time of postfixation |
| Diffuse staining; caused by tissue damage | Carefully prepare tissue sample to avoid tissue damage |
| Insufficient penetration of detection reagents | Prepare thinner sections |
| Different fixatives affect tissue antigen differently | Optimize the pH, incubation time and temperature |
Antigen unmasking is a necessary step prior to antibody labeling, as the tissue fixation process typically causes cross-linking of proteins. This is more common in formalin fixation due to the nature of the chemistry, but can easily be reversed through heat (the most commonly used method), simple buffer treatment or protease digestion. Treatment choice depends on the way the epitope configuration needed for antibody detection. This step re-exposes the epitopes on the antigen and allows antibody binding.
Lab Vision PT Module Deparaffinization and Heat-Induced Epitope Retrieval Solutions (100X)
Lab Vision Protease XXV for Enzyme-Induced Epitope Retrieval
Lab Vision Pepsin Solution for Enzyme-Induced Epitope Retrieval
eBioscience IHC Antigen Retrieval Solution - High pH (10X)
eBioscience IHC Antigen Retrieval Solution - Low pH (10X)
| Possible reasons | Tips |
|---|---|
Antigen is still masked due to protein crosslinking caused during fixation step |
Use proper antigen unmasking method, preferably via microwave; a pressure cooker can also be used, but the use of a water bath is not recommended |
Blocking endogenous enzymes and antibodies within the tissue is a critical for minimizing background staining and reducing false positive staining. This is typically achieved by incubating samples with a buffer that blocks the non-specific sites to which the primary or secondary antibodies may otherwise bind.
eBioscience IHC /ICC Blocking Buffer - Low Protein
eBioscience IHC/ICC Blocking Buffer - High Protein
Avidin/Biotin Blocking Kit
CAS-Block Histochemical Reagent
Blocker FL Fluorescent Blocking Buffer
| Possible reasons | Tips |
|---|---|
| Interference from endogenous peroxidases or phosphatases | Quench with 3% H2O2 in methanol or water or use a kit (i.e., Thermo Scientific Peroxidase Suppressor) prior to using antibodies; use enzyme inhibitors for alkaline phosphatases |
| Interference from endogenous biotin activity | Block using the avidin/biotin blocking reagent before incubating the primary antibodies |
| Interference from endogenous enzymes | Drain the excess buffer instead of washing the tissue sample prior to the addition of antibodies |
When selecting a primary antibody, ensure it has been tested in immunohistochemistry applications. The Invitrogen antibody portfolio comprises over 36,000 high-quality antibodies optimized and tested for IHC. Primary antibodies are available unconjugated (for indirect methods) or in conjugated format for direct detection methods or for multiplexing. Secondary antibodies are also available in a range of conjugates for colorimetric/chromogenic or fluorescence-based IHC.
In addition to antigen detection, cellular processes can also be analyzed in tissue sections prepared for immunohistochemistry. The use of Click chemistry, which combines EdU labeling with colorimetric detection, allows direct detection of cell proliferation in fixed tissues with conventional colorimetric stains. Additionally, the use of Click chemistry with TUNEL labeling allows colorimetric detection of fragmented DNA that occurs during apoptotic events.
Click-iT EdU Colorimetric IHC Detection Kit
Click-iT TUNEL Colorimetric IHC Detection Kit
| Possible reasons | Tips |
|---|---|
| Primary and secondary antibodies have lost their activity | Check antibody for storage conditions, contamination, change in pH or multiple freeze/thaw cycles |
| Primary antibody doesn’t work in IHC application | Make sure antibodies are listed as suitable for use in IHC application |
| Not enough antibody to label available target protein in tissue | Use adequate antibody dilution buffer and proper titration according to the data sheet recommendations |
| Insufficient primary antibody binding | Increase antibody concentration or increase incubation period to overnight at 4°C |
| Primary antibody has not been validated for all types of tissue preparation (fixation method) | Confirm IHC functional application is listed for the primary antibody and the type of IHC you plan to perform (formalin/PFA, fresh, frozen, etc.) |
| Improper species/isotype for protein target or primary antibody (if using a secondary) | Confirm compatibility of primary and secondary antibodies (species and isotype compatibility) |
| Loss of fluorescence signal | If performing fluorescent IHC, ensure the secondary Ab is stored in the dark to prevent degeneration of signal |
| The protein of interest might not be present in the tissue | Check for protein localization and run a positive control |
| The protein of interest is not abundant in the tissue | Amplify the signal using a biotin-conjugated secondary antibody and conjugated streptavidin |
| Phosphorylated protein, requires specialized phosphorylation antibody incubation conditions | Confirm posttranslational modification of target protein and check antibody-specific experimental conditions |
| The epitope might have been modified by the antigen retrieval method used and the antibody does not recognize it | Use a different antigen retrieval method (heat and pH6 or pH9 buffer, enzymatic method, etc.) |
| Protein of interest is a nuclear protein, and is unavailable to be detected by the antibody | Use Triton-X in the blocking buffer and the antibody dilution buffer to permeabilize the membranes |
| High concentration of secondary antibody can reduce antigen detection | Troubleshoot by titering concentration to check signal strength |
| Standard secondary antibodies directly conjugated to HRP may not provide enough signal amplification | Use polymer-based detection reagents or avidin/biotin–based detection systems |
| PBS buffer contains bacteria (contaminated), which can damage phospho sites on proteins | Make certain all buffers are sterile/clean and fresh; visually inspect for clarity |
| Enzyme-substrate reaction is impaired | Do not use buffers with sodium azide in the presence of HRP, and be aware that DI water can contain peroxidase inhibitors that affect enzyme activity |
| Incorrect/non-specific binding | Increase incubation period and/or change blocking agent |
| Possible reasons | Tips |
|---|---|
| Non-specific binding of primary antibodies; if the concentration is too high it elicits high-background and non-specific binding | Use higher dilution of primary antibodies and titrate to optimize concentration |
| Non-specific binding of secondary antibodies | Reduce by treating the tissue with normal serum from the same species as the secondary antibody or use Invitrogen pre-adsorbed, affinity purified secondary antibodies |
| Using antibodies on same species (i.e., mouse antibodies on mouse tissues) | Treat the tissue with mouse on mouse blocking reagent before incubating with primary antibody |
| High incubation temperatures | Decrease temperature or use 4°C incubation In fluorescent IHC |
| Formalin or paraformaldehyde fixatives cause autofluorescence in the green spectrum in fluorescent IHC | Use a fluorophore in the red spectrum or the infrared range (if IR detection system is available) |
| Non-specific binding of polyclonal antibody | Use a monoclonal primary antibody instead of a polyclonal to reduce cross-reactivity |
| Concentration of antibody is too high, causing non-specific binding | Decrease the concentrations of the primary and/or secondary antibodies to reduce nonspecific binding |
| Substrate concentration too high | Reduce the substrate incubation time and dilute the substrate further; choose a different enzyme/substrate pair if your first choice doesn’t work |
Thermo Fisher Scientific offers some of the most innovative microscopy systems for obtaining images of tissues stained by colorimetric or fluorescent labeled antibodies.
| Possible reasons | Tips |
|---|---|
| Light source/path is not set properly | Activate light source or adjust light path |
| Focus not set properly | Adjust focus |
| Filters not properly adjusted | Contact tech support for assistance with EVOS FL Auto 2 Imaging System |
| Staining did not work | Be sure to run a positive control to confirm staining method worked and check method carefully for missing or incorrectly executed steps; contact tech support for further assistance |
| Possible reasons | Tips |
|---|---|
| Objective lens not clean | Clean objective lens properly before visualization |
| Optical aberration of image | Check thickness of cover slips |
| Emission spectrum overlap in fluorescent IHC samples | Choose filter sets appropriate for the label used and choose fluorophores from separate areas of spectrum |
| Incompatibility between chromogen and mounting media leads to signal erosion | Check compatibility between substrate and mounting media; use aqueous mounting media with enzymatic and fluorescent labels or organic mounting media with enzymatic labels only; use DAB substrate with non-aqueous mounting medium and use aqueous mounting media in multiplex analysis |
| Incompatibility of mounting media and coverslip refractive index | Match the refractive index (RI) of the mounting media and the glass (RI= 1.52) as much as possible for tissue transparency and image quality; different types of mounting media have different RIs |
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