Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques to image discrete components in tissues by using appropriately-labeled antibodies to bind specifically to their target antigens in situ. IHC makes it possible to visualize and document the high-resolution distribution and localization of specific cellular components within cells and within their proper histological context. While there are multiple approaches and permutations in IHC methodology, all of the steps involved are separated into two groups: sample preparation and sample staining.

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Introduction

History

The principles of IHC have been known since the 1930s, but it was not until 1942 that the first IHC study was reported. Coons et al. (1942) used FITC-labeled antibodies to identify Pneumococcal antigens in infected tissue. Since then, major improvements have been made in tissue fixation and sectioning methods, antigen/epitope retrieval, antibody conjugation, immunostaining methods and reagents, as well as microscopy itself.   As a result, IHC has become a routine, but essential tool in diagnostic and research laboratories.

Applications

IHC is used for disease diagnosis, biological research, and in drug development.  For example, using specific tumor markers, physicians use IHC to diagnose if a tumor is benign or malignant, to determine its stage and grade, and to identify the cell type and origin of a metastasis in order to find the site of the primary tumor.  A variety of other non-neoplastic diseases and conditions are diagnosed using IHC as a primary tool or as a confirmatory procedure.  In a research context, IHC can be used alone or in conjunction with other analytical techniques to study, for example, normal tissue and organ development, pathological processes, wound healing, cell death and repair, and many other fields.  IHC is also used in drug development to test drug efficacy by detecting either the activity or the up- or down-regulation of disease markers in the target tissues and elsewhere.

Traditional IHC is based on the immunostaining of thin sections of tissues attached to individual glass slides.  Multiple small sections can be arranged on a single slide for comparative analysis, a format referred to as a tissue microarray. 

Typically, IHC slides are prepared, processed, and stained manually or in small groups.   However, current technology provides automation options for high-throughput sample preparation and staining. Samples can be viewed by either light or fluorescence microscopy, and advances in the last 15 years have improved our ability to capture images, quantitate multiparametric IHC data, and increase the collection of that data through high content screening.  Below are some striking examples of IHC staining results obtained with Thermo Scientific Invitrogen antibodies and other IHC reagents.

Detection of HDAC4 in human skin by IHC. Chromogenic IHC was performed on thin sections of human skin obtained from biopsies.  The sections were stained either with a rabbit polyclonal antibody against HDAC4 (Cat. No. PA1-863) or without this antibody (the negative control).  HDAC4 detection was performed using a biotinylated anti-rabbit IgG secondary antibody and streptavidin-Horseradish peroxidase (HRP), followed by colorimetric detection using DAB. The sections were then counterstained with hematoxylin and mounted under coverslips.  In the left hand panel, above the HDAC4 antigen is stained brown by the precipitated DAB reaction product.  The control section on the right is not stained brown because no anti-HDAC4 primary antibody was used. Only the blue hematoxylin counterstaining can be seen. 

IHC detection of cytokeratin 18 in human colon carcinoma tissue by immunofluorescence.  The sections were incubated with a biotinylated anti-cytokeratin 18 antibody and then detected using a Thermo Fisher streptavidin-DyLight 633 conjugate (Cat. No. 21844, red fluorescence).  Thermo Fisher Hoechst stain (e.g. Cat. No. 33342) was used to counterstain the cell nuclei (blue fluorescence). 

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Five steps to publication-quality images

To learn about concrete measures to improve your IHC results, download this free guide. 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:

  1. Sample preparation
  2. Antigen retrieval
  3. Background blocking
  4. Target detection
  5. Sample visualization

Sub-optimal IHC staining can be a problem; however taking steps to optimize the IHC workflow can improve experimental outcome.

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Sample preparation

While using the right antibodies to target the correct antigens and amplify the signal is crucial for optimal visualization, complete preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes.

Tissue collection and perfusion

Human and animal biopsies, or whole organs, are collected for preservation and IHC analysis, depending on the requirements of the researcher. Tissue must be rapidly preserved to prevent the breakdown of cellular protein and degradation of the normal tissue architecture. Often, the tissue is perfused in vivo or in vitro, or simply rinsed free of blood, prior to fixation/preservation.  The goal is to remove blood-derived antigens that may interfere with the detection of target antigens. Tissue perfusion is performed on anesthetized animals by using a peristaltic pump to exsanguinate the animal and rinse the vasculature with sterile saline to remove all blood components from the entire animal or even the desired organ(s). The target organ or tissue can then be collected and fixed prior to IHC.

Tissue fixation

Most tissue fixatives chemically crosslink proteins and/or reduce protein solubility, which can mask target antigens during prolonged or improper fixation. Therefore, the right fixation method must be optimized based on the application and the target antigen to be stained.

The most common fixative is formaldehyde (formalin), a semi-reversible, covalent crosslinking reagent that can be used for perfusion or immersion fixation for any length of time, depending on the level of fixation desired.  Tissues fixed in formaldehyde are typically embedded in paraffin wax to permit sectioning and further processing (see below).  Such tissues and the sections cut from them are often referred to as formalin-fixed and paraffin-embedded or FFPE.  Although formaldehyde is the most commonly used fixative, many other fixatives can also be used (e.g. acetone, methanol).  Generally, use of these alternative fixatives depends on how the target antigens react to fixation in the first place.  Below is another representative example of IHC localization of an antigen, p21 in an FFPE section from a human colon cancer specimen.

Detection of p21 in human lung colon carcinoma by IHC.  IHC staining for p21 in a formalin fixed paraffin embedded (FFPE) section of human colon carcinoma using a monoclonal antibody (Cat. No. MA1-19271) as the primary antibody and an anti-mouse IgG-HRP conjugate as the secondary antibody.  The brown precipitating HRP substrate DAB was used.  Prior to staining, (heat-induced epitope retrieval (HIER) was performed in 10 mM citrate buffer. 

 


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Tissue embedding

Formalin-fixed tissue samples are usually embedded in paraffin to maintain their natural shape and tissue architecture during long-term storage and to facilitate sectioning prior to IHC.   Such samples and the sections prepared from them are usually referred to as formalin-fixed, paraffin-embedded (FFPE) materials.

Samples which are too sensitive for either chemical fixation or the solvents used to remove the paraffin, can be encased in a cryogenic embedding material and then snap-frozen in liquid nitrogen.   Thin slices of these frozen tissue samples are sectioned on a cryostat (freezing microtome), transferred to slides, and then dried to preserve morphology.  Such sections are referred to as frozen or cryosections.

Sectioning and mounting

Paraffin wax is the most commonly used embedding medium for routine histological applications, and formalin-fixed, paraffin-embedded (FFPE) sections produce satisfactory results for detecting most tissue antigens using standard antigen retrieval techniques. However, some antigens are destroyed during routine fixation and paraffin embedding—in which case, frozen tissue sectioning becomes the method of choice. The disadvantages of frozen sectioning include, but are not restricted to these limitations: poor morphology, decreased resolution at high magnifications, and special storage needs.

FFPE tissues are usually cut into sections as thin as 4 to 5 μm with a microtome. These sections are then mounted onto glass slides that are coated with a tissue adhesive. This adhesive is commonly added by surface-treating glass slides with 3-aminopropyltriethoxysilane (APTS) or poly-L-lysine, both of which leave amino groups on the surface of the glass to which the tissue adheres. In the past, and now, if necessary, slides can be coated with actual adhesives, including gelatin, egg albumin or even Elmer's glue. After mounting, the sections are dried in an oven or microwaved in preparation for de-paraffinization.

Frozen sections are cut using a pre-cooled cryostat and mounted to adhesive-coated glass slides. These sections are often dried overnight at room temperature and are usually post-fixed by immersion in pre-cooled (-20°C) acetone, fresh paraformaldehyde, or formaldehyde/formalin at ambient temperature.  The drying step is sometimes skipped depending on the target antigens and tissue being used. In the following IHC example, the protein, VEZF was detected in human brain tissue. 

Chromogenic IHC staining of a paraffin section of human brain. Tissues were processed and probed with PA541131, a rabbit anti-VEZF polyclonal primary antibody.  An anti-rabbit IgG secondary antibody labeled with HRP and the red-precipitating HRP substrate 3-amino-9-ethylcarbazole (AEC) were used for detection.  The red colored regions and fibers represent the locations of VEZF. 

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De-paraffinization and epitope (antigen) retrieval

The paraffin in FFPE sections must be completely removed before IHC staining.   If de-paraffinization is not complete, the target antigens will be obscured and the antibodies will be unable to react with them.  In fact, paraffin's hydrophobicity actually repels aqueous solutions containing the IHC staining reagents.  Flammable, toxic, and volatile organic solvent xylene has traditionally been used to de-paraffinize FFPE slides, although xylene-free de-waxing alternatives are now available. 

Formaldehyde fixation generates methylene bridges that covalently crosslink proteins in tissue samples. These bridges can mask antigen and/or epitope accessibility and inhibit or prevent antibody binding.  As a result, FFPE sections typically require treatment designed to unmask or retrieve the antigenic epitopes prior to staining.  This is called epitope or antigen retrieval.

Epitope/antigen retrieval is usually performed by heating or boiling the de-paraffinized sections in various buffers at different pH values, which is called heat-induced epitope retrieval or HIER.  Antigens can also be retrieved by digesting the tissue sections with a proteolytic enzyme like pepsin, trypsin, or proteinase K.   If antigen or epitope-specific retrieval conditions are not already documented in the literature or on our antibody data sheet, an effective method must be determined empirically.  It is also necessary to mention that although thorough de-paraffinization is always required prior to IHC staining, antigen or epitope retrieval is not.  In some FFPE tissues, certain individual antigens are not obscured, so a retrieval step is not required prior to staining.

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Quenching/blocking endogenous target activity

Many popular staining approaches depend on biotin and its binding proteins like strept(Avidin) (SA), NeutrAvidin (NA), and avidin (AV).  Also, most detection strategies employ horseradish peroxidase (HRP) or alkaline phosphatase (AP) activity for enzyme-mediated detection of target antigens in the presence of specific substrates.   Thus, inactivating (quenching) or masking endogenous forms of these proteins prevents false positive detection and high background staining. The general strategies include physically blocking or chemically inhibiting all endogenous biotin or enzyme activity, respectively.

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Blocking nonspecific sites

Although antibodies show preferential avidity and affinity for specific epitopes, antibodies may partially or weakly bind nonspecifically to sites on non-antigen proteins that mimic the correct binding sites on the target antigen. In the context of antibody-mediated antigen detection, nonspecific binding causes high background staining that can mask the detection of the target antigen. To reduce background staining in IHC, ICC, and any other immunostaining application, prior to staining, the samples are incubated with a buffer that blocks the non-specific sites to which the primary or secondary antibodies may otherwise bind.  Common blocking buffers include some percentage of normal serum, non-fat dry milk, BSA (bovine serum albumin), gelatin, and one or more gentle surfactants to aid in wetting.  Many commercial blocking buffers with proprietary formulations are available for greater blocking efficiency.

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Sample labeling

Immunodetection

Detecting the target antigen with antibodies is a multi-step process that requires optimization at every level to maximize signal detection.

Both primary and secondary antibodies are diluted into a buffer formulated to help stabilize the antibody, promote its uniform and complete diffusion into the sample, and discourage nonspecific binding. While one diluent may work with one antibody, the same diluent may not work with another antibody, demonstrating the need for optimization for each one.

Rinsing the sample in between antibody applications is critical to remove unbound antibodies and also to remove antibodies that are weakly bound to nonspecific sites. Rinse buffers are usually simple solutions with only a few components, but the right components must be considered to maximize washing efficiency and minimize interference with signal detection.

Antibody-mediated antigen detection approaches are separated into direct and indirect methods. Both of these methods use antibodies to detect the target antigen, but the selection of the best method to use depends on the level of target antigen expression, its accessibility, and the type of readout desired. Most indirect methods employ the inherent binding affinity of strept(avidin) and related proteins for biotin to detect a biotinylated antibody that is bound to the target antigen.  The antigen-bound antibody is then localized by adding an enzyme-conjugated strept(avidin) conjugate which generates an amplified signal when appropriate substrates are added.

IHC target antigens are detected directly through either chromogenic or fluorescent means, and the type of readout depends on the experimental design.  Chromogenic detection is based on antibodies conjugated to enzymes.  Most often, the enzymes used are horseradish peroxidase (HRP) or alkaline phosphatase (AP), which are conjugated to primary or secondary antibodies.  When incubated with appropriate substrates, the enzyme activity leads to the precipitation of insoluble, colored precipitates at the antigen localization site.   Such chromogenic, precipitating substrates include DAB and AEC for HRP, and Fast Red and NBT/BCIP (rarely used) for AP, respectively.   For fluorescence detection, the primary or secondary antibody is conjugated to a fluorophore that is detected by fluorescent microscopy.  An example of chromogenic IHC was presented previously, the following image depicts an example of IHC detection by IF to visualize cytokeratin 18 in human colon carcinoma tissue.

IHC detection of cytokeratin 18 in human colon carcinoma tissue by immunofluorescence.  Sections were incubated with a biotinylated anti-cytokeratin 18 antibody and then detected using a Thermo Fisher streptavidin-DyLight 633 conjugate (21844, red fluorescence).  Thermo Fisher Hoechst stain (e.g. 33342) was used to counterstain the cell nuclei (blue fluorescence).

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Counterstaining

Counterstains provide contrast to the primary stain and can be cell structure-specific. These single-step stains are usually added after antibody staining.  Common counterstains include hematoxylin, eosin, nuclear fast red, methyl green, DAPI, and Hoechst fluorescent stain. The following representative example, Hoechst fluorescent dye was used as a counterstain for IHC detection of the protein, vimentin. 

Fixed-tissue staining with Hoechst dye and an antibody.Formalin-fixed paraffin embedded tonsil control tissue was de-paraffinized, subjected to heat-induced epitope retrieval using citrate buffer, and then blocked. The sample was incubated for 30 min at room temperature with mouse anti-vimentin antibody, then for 30 min with orange DyLight Fluor goat anti-mouse secondary antibody. Finally, Hoechst 33342 Solution was added at 1 µg/mL for 5 min. The stained tissue was imaged with the Zeiss Axio Observer .Z1 Microscope (Obj. 20X/0.4NA). 

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Sealing the stained sample

After all staining is completed, the sample should be preserved for archiving purposes and to prevent enzymatic product solubilization or fluorophore photobleaching. Sealing the sample by mounting a coverslip with an appropriate mounting solution (mountant) stabilizes the tissue section and the stain. An antifade reagent should also be included if fluorescent detection was used to prolong fluorescence excitation. The coverslip can then be sealed with clear nail polish or a commercial sealant after the mountant has cured to prevent sample damage.  Mountants with organic and aqueous formulations are commercially available.

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Sample visualization

Once the sections are prepared, the samples are viewed by light or fluorescence microscopy. Depending on the antibody detection method, one can perform confocal microscopy for greater detail and enhanced imaging capabilities. Additionally, samples can be analyzed by high content screening for rapid quantitation and comparison of data from multiple samples.


Recommended reading
  1. Coons, A.A., et al. (1942) J. Immunol. 45, 159-170
  2. Beisker W et al. (1987) Cytometry 8:235–239.
  3. Cowen T et al. (1985) Histochemistry 82:205–208.
  4. Mosiman VL et al. (1997) Cytometry 30:151–156.
  5. Romijn, Herms J. et al. (1999) J Histochem Cytochem 47:229–236.