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Chromogenic in situ Hybridization (CISH) and Silver enhanced in situ Hybridization (SISH)

A simple and practical alternative to FISH that does not depend on expensive fluorescence microscopy instrumentation and expertise has been developed and refined since 2000 and is called Chromogenic in situ Hybridization (CISH).
In CISH, probes are labeled with an antigenic moiety and detected via antibodies conjugated to an enzyme, typically horseradish peroxidase (HRP) or alkaline phosphatase (AP), that catalyzes reactions of chromogenic substrates. The resulting chromogens precipitate at the probe target site and can be detected under a standard bright-field microscope.
In addition to providing a user-friendly economical alternative to FISH, chromogenic hybridization methods have the distinct advantage of enabling visualization of tissue context, including nucleus and cell shape, together with the probe signal on the same image. Unlike the majority of fluorescent detection reagents, chromogenic agents used in most CISH methods are chemically stable and do not fade over time, allowing easy storage and repeated re-examination of samples.
An increasingly popular variation of in situ hybridization techniques employs metallographic detection. Here, the hybridization probe is linked to an enzyme that elicits neutralization and deposition of metal – most commonly silver – out of solution and onto the probe target site (Silver-enhanced in situ Hybridization, SISH). While the resulting stain has only a single color, black, it is generally dense, fine-grained and absolutely stable.
One of the other advantages of SISH testing is that it requires less time than established fluorescent methods. In addition, as with other CISH protocols, results and cell or tissue morphological context can be easily analyzed under a conventional bright field microscope. Moreover, most SISH protocols are well suited to be adapted for running in a fully automated fashion. Although FISH is still the accepted gold standard for the detection of many specific chromosomal abnormalities, a growing number of SISH probe and method alternatives is being developed and validated, especially in the testing for amplified cancer genes.

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