Immunohistochemistry (IHC): the complete guide

Discover your comprehensive guide to immunohistochemistry (IHC). From tissue processing and antibody selection to detection, controls, and troubleshooting, this guide will help boost your research.

Contents

• Tissue processing, fixation, and sectioning
• Antigen retrieval
• Blocking proteins
• Blocking endogenous enzymes
• The primary antibody
• Detection and amplification systems
• Multi-color IHC
• Counterstaining
• Controls for IHC
• Troubleshooting

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.

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IHC RESOURCES

• Kits and reagents
• Primary antibodies for IHC
• Secondary antibodies for IHC
• Our free online training on IHC

Tissue processing, fixation, and sectioning

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.

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Antigen retrieval

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.

IHC of human kidney tissue labeled with rabbit monoclonal anti-ZO1 (ab221547). Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9.0 (ab93684).

Table 2. Primary methods of antigen retrieval.

ANTIGEN RETRIEVAL RESOURCES

• Buffers for antigen retrieval
• Protocol for antigen retrieval

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Blocking proteins

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

• Normal serums for blocking
• Buffers, reagents, and kits for blocking

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Blocking endogenous enzymes

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.

Peroxidase blocking

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.

Alkaline phosphatase blocking

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.

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The primary antibody

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.

Direct vs indirect detection

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.

• Direct detection

  • Suitable for studying highly expressed antigens
  • No need for additional incubation step with a secondary (and therefore eliminates any potential background staining from a secondary)
  • Increased flexibility in the design of multicolor experiments

• Indirect detection

  • Suitable for all antigens
  • The signal may be amplified further with various methods (and is discussed in the following sections)
  • Requires additional blocking steps and controls

IHC RESOURCES

• IHC staining protocol for paraffin, frozen, and free-floating sections

Choosing a 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?

Antibody specificity

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

• Signal-to-noise ratio: the antibody may bind the correct protein, but also have some noise and should be rejected as having a poor signal-to-noise ratio
  • Particularly relevant if KO is not available and you are determining specificity on localization alone
• Staining patterns that are consistent with known localization of the protein of interest in control cells or tissues
• Lack of staining in tissues or cells known not to express the protein.
• Recognition of a single band in western blotting

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.

Clonality

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 limitations.

Table 3. The advantages and limitations 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.

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Detection and amplification systems

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.

Chromogenic vs fluorescent detection

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^®).

• Chromogenic

  • Biotinylated secondary antibodies and streptavidin-HRP can further amplify the signal in an ABC method. Alternatively, you can use a modern HRP-polymer secondary antibody.
  • Some precipitates are photostable (HRP/DAB is very photostable, but HRP/AEC fades in sunlight), potentially allowing storage of the slides for many years.
  • Only requires a standard brightfield microscope.
  • The enzyme/chromogen precipitate is deposited over a wider area than photons from a fluorescent source, which can affect one’s ability to interpret the results.
  • The procedure is generally longer as it includes more incubation and blocking steps than fluorescent methods – however, this isn’t always the case, depending on which amplification system you use.
  • Quantification is generally more difficult due to enzymatic amplification.

• Fluorescent

  • Useful to visualize multiple antigens simultaneously with multiple fluorophores excited at different wavelengths (multiplexing).
  • Better, higher-resolution image quality.
  • The signal can be amplified by using HRP antibody labels with tyramide-dyes to deposit fluorescent dyes at the site of antibody staining.
  • More amenable to signal quantification.
  • Susceptible to auto-fluorescence, particularly with formaldehyde fixation.
  • Faster experimental procedure.
  • Requires more expensive imaging equipment.
  • Fluorophores less stable over time.

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.

Enzymes and chromogens

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.

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

• Detection kits for IHC
• Secondary antibodies for IHC
• Chromogen kits for IHC

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Multi-color IHC

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

• Alexa fluor-conjugated secondary antibodies for mIHC
• Dye-conjugated primary antibodies for mIHC

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Counterstaining

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.

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.

COUNTERSTAINING RESOURCES

• Counterstains and special stains for immunohistochemistry

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Controls for IHC

Run proper controls so that you can confirm the validity of your staining pattern and exclude experimental artifacts.

You should include several different positive and negative controls and maintain a detailed experimental record to ensure consistent performance.

Antigen (tissue) controls

• Positive control: a section from a tissue known to express the protein of interest.

• Negative control: a section from a tissue known not to express the target antigen.

• Endogenous tissue background control: a section from the tissue that doesn’t have a primary antibody applied. Certain tissues have inherent properties that result in background staining, which could affect the interpretation of results. For example, certain tissues contain endogenous fluorescent molecules that could be confused for positive staining during fluorescent IHC. The tissue should be checked under the microscope to ensure that there is no endogenous background.