Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method.
The indoxyl-tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections from the same livers. Addition of polyvinyl alcohol to the incubation medium greatly improved the localization of the final reaction product in cryostat sections. In polyacrylamide films, the formazan production specifically due to alkaline phosphatase was highest when using a medium containing 100 mM Tris-HCl buffer, pH 9.0, 0.2-1.0 mM substrate, 0.32 mM 1-methoxyphenazine methosulphate, 10 mM MgCl2, 5 mM sodium azide and 1 mM tetranitro BT. For the incubation of cryostat sections in the presence of polyvinyl alcohol, the same medium could be used but the optimum concentrations of substrate and tetranitro BT appeared to be 1-2 mM and 5 mM respectively. The test minus control reaction was specific for alkaline phosphatase activity and could be inhibited completely with tetramisole. The test minus control reaction was linear with time up to 30 min with model films and up to 15 min with cryostat sections. The formazan production was also linear with the amount of homogenate incorporated in model films and with section thickness up to 18 micron and therefore, the reaction obeyed the Beer-Lambert law. Variation of the substrate concentration yielded a KM of 0.05 mM for aqueous media and a KM of 0.55 mM for polyvinyl alcohol-containing media.(ABSTRACT TRUNCATED AT 250 WORDS)
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D005629:Frozen Sections; D006651:Histocytochemistry; D007211:Indoles; D008099:Liver / Q000201:enzymology; D051381:Rats; D013053:Spectrophotometry; D013194:Staining and Labeling; D013778:Tetrazolium Salts
D007211:Indoles; D013778:Tetrazolium Salts; C028130:tetranitrotetrazolium blue; C034082:indoxyl; D000469:Alkaline Phosphatase