5-bromo-6-chloro-3-indolylphosphate p-toluidine salt(CAS 6769-80-8) a New Chromogens for Alkaline Phosphatase Histochemistry

     The two chromogens most commonly used today for detecting alkaline phosphatase (ALP) in immunocytochemistry are 5-bromo-4-chloro-3-indolyl phosphate-Nitroblue tetrazolium (BCIP-NBT),and Fast Red-naphthol. Each of these reagents has its own advantages and disadvantages. BCIP-NBT yields an intense blue product that is water- and ethanol-insoluble. However, its blue color does not contrast well with conventional hematoxylin counterstaining. Fast Red-naphthol contrasts well with hematoxylin-stained nuclei, but because it is ethanol-soluble it cannot be used with regressive hematoxylin stains or routine alcohol dehydration and xylene- based mounting media, which are most commonly used and provide the best optical solution.

     We report here the use of two new chromogens for ALP, salmon and magenta phosphate (6-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indolyl phosphate p-toluidine salt,respectively), which have been synthesized and are sold by YACOO Chemicals  (No.17 jinghai road suzhou, china). These reagents have the advantageous attributes of both BCIP-NBT and Fast Red for use in ALP detection in immunocytochemistry. Specifically, these

    reagents produce distinct but relatively pale pink and magenta reaction products, respectively, which are ethanol-insoluble. In addition to these advantages for single antigen detection, both of these colors are easily distinguished from the brown horseradish peroxidase-diaminobenzidine (HRPDAB) reaction product, and are therefore ideal for use in double-immunostaining techniques, especially when distinct nuclear staining with hematoxylin is required. We present here a triple-staining procedure for simultaneous detection of two antigens, using monoclonal antibodies (MAb) with hematoxylin counterstaining.