In situ hybridization (ISH) on routine formalin-fixed paraffin-embedded (FFPE) tissues faces several technical challenges. It has not always been easy to consistently obtain satisfactory ISH results on FFPE tissue sections. The major obstacle is the permeability of the section to the probes and detection reagents. The method that gave the most consistent results in our hand uses microwave to heat slides in an EDTA buffer to a temperature close to boiling for 15 min. The step is similar to the heat-induced antigen-retrieval step that has been popular in the immunohisto-chemistry laboratories. The sections were then digested by pepsin (Zymed Laboratories, South San Francisco, CA) or proteinase K (DAKO Co., Carpinteria, CA). For the ease of use and consistency of results, we recommend the use of commercially available, ready-to-use digestion agents. The amount of time required for the digestion varies according to the type of tissues and method of fixation; consistency is essential so that the result can be easily reproduced.

There are several difficulties of using traditional fluorescent in situ hybridization (FISH) on FFPE sections. The autofluorescence of formalin-fixed tissues often obscures the real fluorescence signals. Tissue morphology is difficult to appreciate under dark-field microscopy at high magnification. Moreover, FISH slides cannot be stored or archived for longer periods of time. We demonstrated here the use of chromogenic in situ hybridization (CISH) for the study of genomic alteration in cancer on FFPE sections. The technique allows ISH data to be interpreted using conventional bright-field microscope.

The detail protocols for two experiments were shown. Although commercial kits are available for both experiments, more detail information was given; thus readers can use it as model to develop protocols for their own experiments. The first experiment uses two-color CISH to examine the translocation of MYC gene on chromosome 8 in Burkitt lymphoma. The second example demonstrates combined IHC and CISH for HER2 protein expression and gene amplification in breast cancer.

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