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Immunohistochemistry (IHC) uses antibodies to detect the location of proteins and other antigens in tissue sections. The antibody-antigen interaction is visualized using either chromogenic detection with a colored enzyme substrate, or fluorescent detection with a fluorescent dye.
Although less quantitative than assays such as western blotting or ELISA, IHC gives invaluable information about protein localization in the context of intact tissue. Protein expression patterns are tremendously valuable for pathologists and as diagnostic tools.
Essential to a successful IHC experiment is a robust, optimized, and reproducible staining regimen that makes use of high-quality, specific reagents.
Tissue fixation preserves antigens and prevents the autolysis and necrosis of harvested tissues. Embedding tissue provides support during sectioning and makes sections more robust.
The first decision when planning an IHC study is how to prepare the tissue sections. The most common method uses paraffin embedding. Frozen sections and floating sections are other options – each method has advantages and limitations (Table 1).
Sample fixation is key to tissue processing and is critical to prevent the degradation of antigens, cells, and tissue. Solutions of 10% buffered formalin and 4% formaldehyde (also called paraformaldehyde) are typical fixatives – these are near identical; formalin is a 40% solution of formaldehyde.
It is critical to fix or freeze samples quickly and thoroughly after harvesting and to ensure that samples are not too large to fix completely or freeze quickly.
Table 1. Paraffin vs freezing vs floating for IHC.
Pre or post-sectioning: formaldehyde, methanol, ethanol, or acetone
Tissue dehydrated and cleared before adding paraffin (pre-heated to 60oC) and left overnight.
Tissue frozen by immersion in liquid nitrogen, isopentane or by burying the sample in dry ice.
Snap-freezing is common when detecting post-translation modifications such as phosphorylation.
Embedding not required.
Multiple years at room temperature.
1 year at -80°C (longer at -190°C).
In cryoprotectant at -20°C, or short-term in PBS + azide at 4°C.
Easy to handle without damaging the section.
Preserves enzyme function and antigenicity.
Shorter protocol (lengthy fixation step usually not required).
Used with thicker sections (>25 µm): allows greater analysis of the 3D structure of the tissue.
Over-fixation can mask the epitope – increased requirement for antigen retrieval.
Lengthy processing: eg gradual dehydration in alcohol series and xylene to allow paraffin penetration.
Formation of ice crystals may negatively affect tissue structure if tissues are not frozen rapidly.
Sections produced are often thicker than paraffin sections, increasing the potential for lower resolution and poorer images.
May need to block active endogenous enzymes.
More challenging to image smaller structures and individual cells.
Additional tissue clearing methods, such as CLARITY, may be required to reduce light scattering and image thicker sections.
Perform antigen retrieval on formaldehyde-fixed tissue sections to expose antigenic sites and allow antibodies to bind.
Formaldehyde fixation results in protein cross-linking (methylene bridges), which masks epitopes and can restrict antigen-antibody binding. Antigen retrieval methods (Table 2) break these methylene bridges and expose antigenic sites, allowing antibodies to bind.
Frozen tissue sections are often not robust enough to be used with antigen retrieval without damaging the section. Many people tend to avoid using formaldehyde fixatives with frozen sections (or they are used with greatly reduced exposure time), thereby removing/reducing the need for antigen retrieval.
Table 2. Primary methods of antigen retrieval.
Heat-induced epitope retrieval
Proteolytic-induced epitope retrieval
Gentler epitope retrieval and more definable parameters.
Useful for epitopes that are difficult to retrieve.
pH 6 buffers are often used, but high pH buffers are widely applicable. Optimal pH must be determined experimentally.
Typically pH 7.4.
Depends on the pH required for the target antigen.
Popular buffer solutions include sodium citrate, EDTA, and Tris-EDTA.
Neutral buffer solutions of enzymes such as pepsin, proteinase K or trypsin.
Heating with microwaves can result in uneven epitope retrieval due to hot and cold spots. Rigorous boiling can lead to tissue dissociation from the slide.
Enzymatic retrieval can sometimes damage the morphology of the section – concentration and timing need to be optimized.
ANTIGEN RETRIEVAL RESOURCES
Block with sera or a protein to prevent non-specific antibody binding and reduce background and potentially false positive results.
Blocking with sera or a protein blocking reagent is essential to prevent non-specific binding of antibodies to tissue or Fc receptors (a receptor that binds the constant region (Fc) of an antibody).
A serum matching the species of the secondary antibody is an excellent blocking reagent. Proteins such as bovine serum albumin (BSA) or casein can be used to block non-specific antibody binding.
We recommend blocking endogenous biotin when using an avidin/biotin-based detection system since endogenous biotin is present in many tissues, particularly in the kidney, liver, and brain. You first block before incubating the tissue with avidin and then incubate with biotin to block additional biotin binding sites on the avidin molecule.
If using a primary antibody raised in the same species as your sample (eg mouse antibody on mouse tissue), then block with a F(ab) fragment of a secondary antibody against that species. The F(ab) fragment binds to, and saturates, any endogenous antibodies in the tissue section, blocking binding of the secondary antibody. However, this F(ab) approach does not produce a complete block and does leave some background.
PROTEIN BLOCKING RESOURCES
For enzymatic detection methods, block endogenous enzymes so as not to confound your results. Consider blocking endogenous enzymes after incubating with your primary antibody, as treatments like H2O2 can damage epitopes and affect binding. If your antibody is an HRP primary conjugate, then this block needs to be done before the addition of the primary antibody.
Chromogenic detection methods usually use an enzyme conjugated to a secondary antibody to visualize antibody localization. If the enzymatic activity is also endogenous to your tissue sample, it’s important to block the endogenous enzymes before the detection step.
When using horseradish peroxidase (HRP)-conjugated antibodies for detection, non-specific or high background staining may occur due to endogenous peroxidase activity. Incubate tissues with 3,3'-diaminobenzidine (DAB) substrate before primary antibody incubation to check for endogenous peroxidase activity. If the tissues turn brown, endogenous peroxidase (found in red blood cells, for example, which are generally within vessels within the tissue) is present and you require a blocking step. Hydrogen peroxide (H2O2) is the most common peroxidase blocking agent.
Endogenous alkaline phosphatase (AP) can produce high background when using an AP-conjugated antibody for detection. Tissue can be tested for endogenous AP by incubating with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride (BCIP/NBT); if a blue color appears, then endogenous AP is present, and blocking is necessary. There are several alkaline phosphatase inhibitors available, including levamisole hydrochloride and tetramisole hydrochloride.
A critical decision when designing an IHC experiment is primary antibody selection since successful immunostaining relies on your primary antibody specifically binding the target antigen.
Before choosing your primary, you need to consider whether you plan to use direct or indirect detection methods. The antibody is detected either directly, through a label that is directly conjugated to the primary antibody, or indirectly, using a labeled secondary antibody raised against the host species and antibody type and subtype of the primary antibody.
Main points to consider: what species is it raised in? Does it bind the intended protein? And has it been shown to work in your application before?
The most conclusive demonstration of antibody specificity is lack of staining in tissues or cells in which the target protein has been knocked out. Other indicators are
ICC image of knockout testing for our Ki67 antibody in wildtype (top) and knockout HAP1 cells (bottom). Green is anti-Ki67 [EPR3610] (ab92742)with goat-anti rabbit IgG (Alexa Fluor® 488) (ab150081), red is anti-alpha-tubulin [DM1A] (Alexa Fluor® 594) (ab195889), and blue is nuclear DNA labeled with DAPI.
Prior use in IHC
An antibody that recognizes its target protein in western blotting experiments may not always recognize the antigen in IHC, where the antigen is more likely to be in its native (tertiary 3D) form. An antibody that has been shown to work in IHC is preferable.
Antibody clonality is determined by whether the antibodies come from different B-cells (producing polyclonal antibodies) or from identical B-cells derived from a parent clone (producing monoclonal antibodies). These have distinct advantages and limitation.
Table 3. The advantages and limitation of polyclonal vs monoclonal primary antibodies.
*Antigen/epitope affinity purification makes polyclonal antibodies more specific as a population, especially if the antigen is short, such as a peptide.
For indirect detection, the secondary antibody is critical to successfully visualizing the distribution of your primary antibody.
Unlike direct detection using a labeled primary antibody, the use of secondary antibodies and related detection systems enable signal amplification as more than one secondary antibody molecule binds to each primary.
Your detection methods can be either chromogenic, using secondary antibodies that are enzyme-labeled (eg, HRP, AP), or fluorescent (immunofluorescence) using secondary antibodies that are fluorochrome-labeled (eg, FITC, R-PE, Alexa-Fluor®).
Fluorescent IHC image of NeuN in paraffin-embedded mouse cerebellum tissue sections. Green is anti-NeuN [EPR12763] (ab177487), with goat anti-rabbit IgG conjugated to Alexa Fluor® 488 (ab150097), red is anti-GFAP (ab4674), with goat anti-chicken IgY conjugated to Alexa Fluor® 594 (ab150176). Image by Carl Hobbs, Kings's College London, UK.
Additional factors for consideration in chromogenic detection are the choice of enzymatic and chromogenic substrates. Several different chromogens are available for each detection enzyme (Table 4). HRP-DAB is the most popular combination.
One advantage of chromogens is that you can use them with an organic mounting medium, which tends to have a better refractive index, resulting in sharper images. However, aqueous mediums are faster to use as there is no need to dehydrate the section.
Table 4. Popular enzymes and substrates/chromogens for IHC.
Intense color; permanent
Endogenous peroxidase activity in tissue can lead to false positive staining
DAB + nickel enhancer
Intense color; permanent
Intense color; contrasts well with blue in double staining
Endogenous AP activity in tissue can lead to false positives
IHC staining of paraffin-embedded wild type (A) and GSDMD KO mouse small intestine (B) with anti-GSDMD antibody [EPR20859] (ab219800) and HRP-polymer conjugated secondary antibody used in our micro-polymer IHC detection kits. Tissue kindly provided by Dr. Feng Shao, NIBS.
DETECTION AND AMPLIFICATION RESOURCES
Multiple markers can be immunostained in a single tissue section using multi-color IHC (mIHC).
Traditional chromogenic mIHC relies on each antibody being raised in a different species or of a different isotype. Specific secondary antibodies are then used, with a different chromogen for each marker. However, it is hard to distinguish more than two chromogens on a slide, particularly if any chromogens overlay each other.
Fluorescent mIHC can be easily used with three or more markers. It can be used with fluorescent dye-conjugated primary antibodies however it is more commonly used with dye-conjugated secondary antibodies, due to their extra amplification, and the limited availability of primary dye conjugates. Most fluorescent mIHC is limited to three markers (plus a counterstain) by available fluorescence filter sets, and by the need for each primary antibody to be raised in a different species / have a different isotype.
The most common methods to increase the number of markers further use: a) spectral unmixing microscopes that enable more fluorescent dyes to be distinguished; and b) sequential antibody stripping and staining methods, often with tyramide signal amplification. Other methods such as imaging mass cytometry, rely on generating a pseudo-image.
mIHC permits high-content data to be generated from one tissue section, effectively reducing the amount of tissue required, and allowing the relationship between different markers to be better understood.
Multi-color fluorescent IHC staining of neonatal pancreas in mice using collagen IV (yellow), insulin (green), and glucagon (red) primary antibodies, and Cy2, Cy5 and Texas Red-conjugated secondary antibodies. Image from Miller K et al. PLoS One 4(11): e7739
MULTI-COLOR IHC RESOURCES
Use a counterstain for specific morphologies or structures to aid localization of your primary antibody.
When performing IHC, it is important to use a counterstain, so that you can see where the staining from the antibody is in relation to the cellular structures within the tissue.
The most popular counterstain used with chromogenic IHC staining is hematoxylin, which stains nuclei blue, contrasting with the brown of HRP-DAB. Hematoxylin is 'blued' with a weakly alkaline solution (tap water is sufficient in most areas but this needs to be determined).
In fluorescent IHC the most popular counterstain is the blue nuclear dye DAPI.
In both cases, be sure to choose a counterstain that it is compatible with your staining system and doesn’t interfere with the signals from your reporter labels.
Table 5. Common counterstains and their targets.
Blue to violet
Nuclear fast red (Kernechtrot)
Nuclear yellow (Hoechst S769121)
Nuclear Green DCS1
IHC image of Iron Stain Kit (ab150674) in formalin-fixed-paraffin embedded human liver. Blue is the iron stain, pink is nuclear fast red. Can also be used to identify specific features of the tissue and is sometimes used as a counterstain.
Run proper controls so that you can confirm the validity of your staining pattern and exclude experimental artefacts.
You should include several different positive and negative controls and maintain a detailed experimental record to ensure consistent performance.
IHC staining of select tumors and reactive tissue stained for c-Myc with anti-c-Myc antibody [Y69] (ab32072) from Kluk MJ et al. PLoS One (2012) 7(4): e33813. Burkitt lymphoma with a confirmed MYC translocation is positive control (high), negative control (low) is healthy tonsil.
Here are some of the common problems you are likely to encounter during IHC and possible causes and solutions.
The primary antibody and the secondary antibody are not compatible.
Not enough primary antibody is bound to the protein of interest.
The antibody may not be suitable for IHC procedures as it may not recognize the native (3D) form of the protein.
The primary antibody/secondary antibody/amplification kit may have lost its activity due to improper storage, improper dilution or multiple cycles of freeze/thaw.
The protein is not present in the tissue of interest.
The protein of interest is not abundant in the tissue.
The secondary antibody was not stored in the dark (when performing fluorescence detection).
Deparaffinization may be insufficient.
Fixation procedures may be modifying the epitope that the antibody recognizes.
The antibody cannot penetrate the nucleus, where the protein is located (nuclear protein).
The PBS buffer is contaminated with bacteria that damage the phosphate groups on the protein of interest (phosphorylated proteins).
Blocking of non-specific binding might be absent or insufficient.
The primary antibody concentration may be too high.
Incubation temperature may be too high.
The secondary antibody may be binding non-specifically.
Tissue has not been washed enough; fixative still present.
Endogenous peroxidases are active.
Fixation procedures are causing autofluorescence (if using fluorescence detection).
Too much amplification (indirect technique).
Too much substrate was applied (enzymatic detection).
The chromogen reacts with PBS present in the tissue sample (enzymatic detection).
Permeabilization has damaged the membrane and removed the membrane protein (membrane protein).
Primary/secondary antibody concentration may be too high.
Endogenous peroxidases are active.
The primary antibody is raised against the same species as the tissue stained (eg, mouse primary antibody tested on mouse tissue). When the secondary antibody is applied, it binds to all the tissue as it is raised against that species.
The sections/cells have dried out.
Antigen retrieval methods may be too harsh.
The tissue may have been under-fixed.
Tissue sections are falling off the slide (frozen sections).
Tissue sections are torn or folded, or air bubbles are visible under the sections.
The tissue morphology is poorly resolved.
The tissue has autolyzed.