Description
The application of antibodies and other reagents such as avidin, streptavidin, etc., covalently coupled to calf intestine alkaline phosphatase in immunohistology is well documented. The procedure described in this insert employs a simultaneous capture azo-dye technique, providing the research laboratory a method for precise localization of alkaline phosphatase labeled reagents. Primary aryl amines, when reacted with alkyl nitrites in acid media, form azo compounds. These react with substituted naphthols to produce highly chromogenic insoluble dyes. In this procedure the phosphate ester of 6-bromo-2-hydroxy-3-naphthoic acid (Buffered Substrate Solution) is employed as substrate. Enzymatic hydrolysis, in the presence of hexazotized triaminotrimethyltriphenylmethane (PhThaloRED Solution) results in the formation of a brilliant red reaction product. Endogenous enzyme is eliminated by incorporation of levamisol. It should be noted that a levamisole-resistant alkaline phosphatase has been demonstrated in some malignant cells from serous effusions. Additional blocking measures may be required.
