Slides are processed for immunohistochemistry to detect other markers immediately following color detection of the ISH signal. The immunohis-tochemical probe is visualized with Fast Red (Fig. 1). One potential problem of using this protocol is residual alkaline phosphatase activity from the ISH, which may produce a product with the Fast Red substrate. Controls in which no primary antibody was used during immunohistochemistry show that this is not generally the case. If residual alkaline phosphatase activity is a concern with other probes or reagents it can be inactivated by treatment with 0.1 M glycine HCl, pH 2.2, prior to immunohistochemistry. Occasionally the proteinase K step used in the ISH protocol may destroy the antigen for subsequent immunohistochemical staining. In such cases a different method of antigen retrieval, such as pressure cooking or microwaving can be used instead (5).
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