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DEMONSTRATION OF ACID PHOSPHATASE ACTIVITY USING 1-ACETYL-3-INDOLYL PHOSPHATE AS SUBSTRATE

MASANDO HAYASHI 1

1 Departments of Pathology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215

A simultaneous coupling azo-indoxyl method for the cytochemical demonstration of acid phosphatase activity using p-toluidine salt of 1-acetyl-3-indolyl phosphate is described. A satisfactory staining for the enzyme activity was obtained following incubation of formol-calcium-fixed frozen sections for 30 min at 25°C in a medium containing 1 mM each of the substrate and hexazonium pararosanilin and adjusted to pH 4.5-5.0 with acetate buffer. The distribution of acid phosphatase activity demonstrated by this method was identical with that obtained either by Gomori's technique using beta-glycerophosphate as substrate or by the Barka and Anderson's naphthol AS-BI phosphate-hexazonium pararosanilin method in several tissues of male rats so far examined. However, the adrenal enzyme activity was most prominent in the medulla with 1-acetyl-3-indolyl phosphate and beta-glycerophosphate but it was more marked in the cortex with naphthol AS-BI phosphate. An advantage of using 1-acetyl-3-indolyl phosphate as substrate is that the same compound can be used for comparing azo-indoxyl and lead-salt methods. Effects of phospholipase C and Triton X-100 on staining for acid phosphatase were tested by pretreating fixed rat liver and kidney sections with these agents and incubating them in the medium containing 1-acetyl-3-indolyl phosphate and either hexazonium pararosanilin or lead ions as a coupler. The pretreatment did not change discrete lysosomal staining, as seen in untreated controls, using pararosanilin as a coupler, but greatly modified the staining using lead ions. The results indicate that the preciseness of staining for acid phosphatase with lead-salt method is highly dependent on some lipid material which attracts lead in tissues and that appropriately devised azo dye or azo-indoxyl methods demonstrate enzyme sites more accurately than lead-salt method.

Submitted on November 5, 1970


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