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Immunohistochemistry

Immunohistochemistry (IHC) combines anatomical, immunological and biochemical techniques to identify discrete tissue components by the interaction of target antigens with specific antibodies tagged with a visible label. IHC makes it possible to visualize the distribution and localization of specific cellular components within cells and in the proper tissue context. While there are multiple approaches and permutations in IHC methodology, all of the steps involved are separated into two groups: sample preparation and labeling.

IHC is used for disease diagnosis, drug development and biological research. Using specific tumor markers, physicians use IHC to diagnose a cancer as benign or malignant, determine the stage and grade of a tumor, and identify the cell type and origin of a metastasis to find the site of the primary tumor. IHC is also used in drug development to test drug efficacy by detecting either the activity or the up- or down-regulation of disease targets.

Samples are prepared on individual slides, or multiple samples can be arranged on a single slide for comparative analysis, such as with tissue microarrays. IHC slides can be processed and stained manually, while technological advances now provide automation 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 the ability to capture images, quantitate multiparametric IHC data and increase the collection of that data through high content screening.

IHC target antigens are detected through either chromogenic or fluorescent means, and the type of readout depends on the experimental design. For fluorescent detection, the reporter that the primary or secondary antibody is conjugated to is a fluorophore that is detected by fluorescent microscopy. Chromogenic detection is based on the activites of enzymes, most often horseradish peroxidase (HRP) or alkaline phosphatase (AP), which form colored, insoluble precipitates upon the addition of substrate, such as DAB and NBT/BCIP, respectively.

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