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Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a technique combining anatomical, biochemical and immunological methods to identify specific antigen-antibody interactions in tissue sections. IHC has been used in a wide variety of formats since the 1940's but there are two steps involved in all methods: sample preparation and labeling.

IHC is used in basic research to understand the distribution and localization of cellular markers and differentially expressed proteins in different parts of a biological tissue. IHC is used in the staining of abnormal cells, such as in cancerous tumors, for specific markers of cell proliferation and cell death (both apoptosis and necrosis).

Sample preparation is critical for optimal visualization of antibody-antigen interactions. Samples must be prepared in a manner that maintains cell morphology, including the underlying tissue architecture and also maintains the epitopes of the target antigens. Fixation is used to preserve tissue and common agents include formaldehyde, paraformaldehyde and glutaraldehyde, which all act, to varying degrees, to reversibly cross-link covalent bonds in proteins, but this fixation can sometimes result in the masking of the epitope. Therefore, fixation protocols must be experimentally determined and optimized, based on the tissue and the antibody used for detection.

Sample labeling to visualize an antibody-antigen interaction can be accomplished in a number of ways, including tagging the antibody to a fluorophore such as fluoroscein or rhodamine for viewing under light or confocal microscopy.



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