What Is Immunohistochemistry Used For

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 information technology possible to visualize and document the high-resolution distribution and localization of specific cellular components inside 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.

Introduction

History

The principles of IHC have been known since the 1930s, just information technology was not until 1942 that the first IHC written report was reported. Coonset al. (1942) used FITC-labeled antibodies to identifyPneumococcal antigens in infected tissue. Since and so, major improvements take been made in tissue fixation and sectioning methods, antigen/epitope retrieval, antibody conjugation, immunostaining methods and reagents, as well equally microscopy itself. Equally a result, IHC has become a routine, but essential tool in diagnostic and research laboratories.

Applications

IHC is used for affliction diagnosis, biological research, and in drug development. For example, using specific tumor markers, physicians utilise IHC to diagnose if a tumor is benign or malignant, to decide its phase and grade, and to identify the cell type and origin of a metastasis in gild to observe the site of the master tumor. A variety of other not-neoplastic diseases and atmospheric condition are diagnosed using IHC as a primary tool or as a confirmatory procedure. In a research context, IHC can exist used alone or in conjunction with other analytical techniques to study, for case, 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 upwardly- or downward-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 assay, a format referred to equally a tissue microarray.

Typically, IHC slides are prepared, candy, and stained manually or in small groups. However, current engineering science provides automation options for high-throughput sample preparation and staining. Samples tin be viewed past either light or fluorescence microscopy, and advances in the last 15 years have improved our ability to capture images, quantitate multiparametric IHC information, and increment 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 man pare by IHC. Chromogenic IHC was performed on sparse 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 antibiotic (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 nether coverslips. In the left hand panel, above the HDAC4 antigen is stained brownish by the precipitated DAB reaction production. The control department on the right is not stained brown considering 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 and then detected using a Thermo Fisher streptavidin-DyLight 633 conjugate (Cat. No. 21844, ruby-red fluorescence). Thermo Fisher Hoechst stain (e.g. True cat. No. 33342) was used to counterstain the jail cell nuclei (blue fluorescence).

Five steps to publication-quality images

To acquire nigh concrete measures to improve your IHC results, download this costless guide. Whether you lot 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 starting time time:

Sample preparation
Antigen retrieval
Groundwork blocking
Target detection
Sample visualization

Immunohistochemistry: five steps to publication-quality images

Sub-optimal IHC staining tin can be a problem; withal taking steps to optimize the IHC workflow can meliorate experimental outcome.

Download brochure Find IHC antibodies

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

Tissue collection and perfusion

Homo and creature biopsies, or whole organs, are nerveless for preservation and IHC analysis, depending on the requirements of the researcher. Tissue must be rapidly preserved to foreclose the breakdown of cellular poly peptide and degradation of the normal tissue architecture. Often, the tissue is perfused in vivo or in vitro, or simply rinsed gratis 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 fauna and rinse the vasculature with sterile saline to remove all blood components from the entire animate being or even the desired organ(due south). The target organ or tissue tin and then exist collected and stock-still prior to IHC.

Tissue fixation

Near tissue fixatives chemically crosslink proteins and/or reduce poly peptide solubility, which tin can mask target antigens during prolonged or improper fixation. Therefore, the correct 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 fourth dimension, depending on the level of fixation desired. Tissues fixed in formaldehyde are typically embedded in paraffin wax to let sectioning and further processing (encounter beneath). Such tissues and the sections cut from them are oftentimes referred to as formalin-stock-still and paraffin-embedded or FFPE. Although formaldehyde is the most normally used fixative, many other fixatives can too be used (due east.1000. acetone, methanol). Mostly, use of these alternative fixatives depends on how the target antigens react to fixation in the get-go 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 stock-still paraffin embedded (FFPE) section of human colon carcinoma using a monoclonal antibody (True cat. No. MA1-19271) equally the primary antibiotic and an anti-mouse IgG-HRP conjugate as the secondary antibody. The dark-brown precipitating HRP substrate DAB was used. Prior to staining, (rut-induced epitope retrieval (HIER) was performed in 10 mM citrate buffer.

Tissue embedding

Formalin-fixed tissue samples are usually embedded in paraffin to maintain their natural shape and tissue compages 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, alkane-embedded (FFPE) materials.

Samples which are likewise sensitive for either chemical fixation or the solvents used to remove the alkane series, can exist 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 every bit frozen or cryosections.

Sectioning and mounting

Paraffin wax is the nigh usually used embedding medium for routine histological applications, and formalin-fixed, paraffin-embedded (FFPE) sections produce satisfactory results for detecting nearly tissue antigens using standard antigen retrieval techniques. However, some antigens are destroyed during routine fixation and paraffin embedding—in which example, frozen tissue sectioning becomes the method of choice. The disadvantages of frozen sectioning include, but are non 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 four to 5 μm with a microtome. These sections are then mounted onto glass slides that are coated with a tissue adhesive. This adhesive is unremarkably 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 by, and now, if necessary, slides tin be coated with actual adhesives, including gelatin, egg albumin or even Elmer's gum. After mounting, the sections are dried in an oven or microwaved in preparation for de-paraffinization.

Frozen sections are cutting using a pre-cooled cryostat and mounted to adhesive-coated glass slides. These sections are oft stale overnight at room temperature and are usually mail service-stock-still 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 existence used. In the following IHC example, the protein, VEZF was detected in man encephalon tissue.

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

De-paraffinization and epitope (antigen) retrieval

The methane series in FFPE sections must be completely removed before IHC staining. If de-paraffinization is non complete, the target antigens will be obscured and the antibodies volition be unable to react with them. In fact, methane series's hydrophobicity actually repels aqueous solutions containing the IHC staining reagents. Combustible, toxic, and volatile organic solvent xylene has traditionally been used to de-paraffinize FFPE slides, although xylene-gratuitous de-waxing alternatives are now available.

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

Epitope/antigen retrieval is commonly performed by heating or boiling the de-paraffinized sections in diverse buffers at dissimilar pH values, which is chosen heat-induced epitope retrieval or HIER. Antigens tin 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 adamant empirically. It is also necessary to mention that although thorough de-paraffinization is ever required prior to IHC staining, antigen or epitope retrieval is non. In some FFPE tissues, certain individual antigens are not obscured, and then a retrieval pace is non required prior to staining.

Quenching/blocking endogenous target activity

Many popular staining approaches depend on biotin and its binding proteins similar strept(Avidin) (SA), NeutrAvidin (NA), and avidin (AV). Also, most detection strategies utilise 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.

Blocking nonspecific sites

Although antibodies show preferential ardor 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 groundwork staining that can mask the detection of the target antigen. To reduce groundwork 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 main or secondary antibodies may otherwise demark. Common blocking buffers include some per centum of normal serum, non-fat dry milk, BSA (bovine serum albumin), gelatin, and ane or more gentle surfactants to aid in wetting. Many commercial blocking buffers with proprietary formulations are available for greater blocking efficiency.

Immunodetection

Detecting the target antigen with antibodies is a multi-step process that requires optimization at every level to maximize point 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 bounden. While i diluent may work with one antibiotic, the same diluent may not piece of work with some other antibody, demonstrating the need for optimization for each 1.

Rinsing the sample in betwixt antibody applications is critical to remove unbound antibodies and also to remove antibodies that are weakly jump to nonspecific sites. Rinse buffers are commonly simple solutions with only a few components, just the right components must exist considered to maximize washing efficiency and minimize interference with betoken detection.

Antibody-mediated antigen detection approaches are separated into straight and indirect methods. Both of these methods use antibodies to observe the target antigen, but the pick 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 analogousness of strept(avidin) and related proteins for biotin to detect a biotinylated antibody that is bound to the target antigen. The antigen-leap antibody is then localized by calculation 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 alkali metal 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 Reddish and NBT/BCIP (rarely used) for AP, respectively. For fluorescence detection, the master or secondary antibody is conjugated to a fluorophore that is detected past fluorescent microscopy. An case of chromogenic IHC was presented previously, the following image depicts an instance of IHC detection by IF to visualize cytokeratin 18 in man colon carcinoma tissue.

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

Counterstaining

Counterstains provide contrast to the primary stain and can exist cell structure-specific. These unmarried-step stains are usually added after antibiotic staining. Common counterstains include hematoxylin, eosin, nuclear fast ruddy, methyl green, DAPI, and Hoechst fluorescent stain. The following representative example, Hoechst fluorescent dye was used equally a counterstain for IHC detection of the poly peptide, vimentin.

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

Sealing the stained sample

Later 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 advisable mounting solution (mountant) stabilizes the tissue department and the stain. An antifade reagent should also be included if fluorescent detection was used to prolong fluorescence excitation. The coverslip tin then be sealed with clear nail polish or a commercial sealant after the mountant has cured to forbid sample damage. Mountants with organic and aqueous formulations are commercially bachelor.

Once the sections are prepared, the samples are viewed past 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 past loftier content screening for rapid quantitation and comparing of information from multiple samples.

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