Immunohistochemistry is the most common application of immunostaining. It involves the process of selectively identifying antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo", meaning tissue. Visualising an antibody-antigen interaction can be accomplished in a number of ways, mainly either of the following:

• Chromogenic immunohistochemistry wherein an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction.
• Immunofluorescence, where the antibody is tagged to a fluorophore, such as fluorescein or rhodamine.

Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death Immunohistochemistry is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.

Preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning. A solution of formalin is often used to fix tissue, but other methods may be used.

The tissue may then be sliced or used whole, dependent upon the purpose of the experiment or the tissue itself. Before sectioning, the tissue sample may be embedded in a medium, like paraffin wax or cryomedia. Sections can be sliced on a variety of instruments, most commonly a microtome, cryostat, or vibratome. Specimens are typically sliced at a range of 3 µm-5 μm. The slices are then mounted on slides, dehydrated using alcohol washes of increasing concentrations and cleared using a detergent like xylene before being imaged under a microscope.

Depending on the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigen-retrieval is often necessary, and involves pre-treating the sections with heat or protease. These steps may make the difference between the target antigens staining or not staining.

A great amount of non-specific binding causes high background staining which will mask the detection of the target antigen. Methods to eliminate background staining include dilution of the primary or secondary antibodies, changing the time or temperature of incubation, and using a different detection system or different primary antibody. Quality control should as a minimum include a tissue known to express the antigen as a positive control and negative controls of tissue known not to express the antigen, as well as the test tissue probed in the same way with omission of the primary antibody.

Surgical Pathology and Diagnosis is a peer-reviewed medical journal published by Oxford University Press covering research on the pathogenesis, clinical investigation, medical microbiology, diagnosis, immune mechanisms, and treatment of diseases.

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Eliza Grace

Journal Manager

Journal of Surgical Pathology and Diagnosis