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Two new substrate chromogens for alkaline phosphatase (ALP) detection have been recently synthesized for use in molecular biology research, salmon and magenta phosphate. We show here that these two chromogens have advantageous characteristics for immunocytochemistry as well. Their relatively delicate pink- and magenta-colored products do not mask the colors produced by other staining procedures. In addition, the reaction products of these substrates are insoluble in water, ethanol, and xylene, permitting the use of regressive hematoxylin staining procedures and coverslipping in permanent resin-based media. Most importantly, when these ALP substrates are used in double-label immunocytochemistry in combination with horseradish peroxidase-diaminobenzidine (HRP-DAB) and counterstained with hematoxylin, all three colors can be easily distinguished. An application using these substrates for simultaneous immunocytochemical detection of two monoclonal antibodies of different classes, in combination with hematoxylin staining, is illustrated.

New chromogens for alkaline phosphatase histochemistry: salmon and magenta phosphate are useful for single- and double-label immunohistochemistry.