page_title: Immunohistochemistry
section_title: IHC reporters
hierarchical_section_title: Immunohistochemistry / Sample labeling / IHC reporters
caption_attribution_description: English: Diagram created by Viki Male, 14th September 2005, to illustrate indirect immunohistochemical staining.
context_page_description: 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. Albert Coons conceptualized and first implemented the procedure in 1941.
  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.
context_section_description: Reporter molecules vary based on the nature of the detection method, the most popular being chromogenic and fluorescence detection mediated by an enzyme or a fluorophore, respectively. With chromogenic reporters, an enzyme label reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Reaction with DAB can be enhanced using nickel, producing a deep purple/black staining.
  Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization.
caption: The indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.
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