HISTOCHEMICAL DEMONSTRATION OF ACID PHOSPHATASE BY AN IMPROVED AZO-DYE METHOD

Histochemical demonstration of acid phosphatase by an azo-coupling procedure has been investigated and conditions established whereby acceptable histological localisations without evidence of diffusion artefact may be secured. These include catalysis of the coupling reaction and retarding of the rate of enzymic liberation of free naphthol. More rapid coupling is achieved in a medium of pH 5.5-pH 6.0 by the use of a high concentration of diazonium salt, by incubation at 37°C. and by the presence of a quinoline derivative, percain. Liberation of free naphthol occurs relatively slowly at the non-optimal pH range employed and is further restricted by lowering of substrate concentration; in this manner local overloading of the coupling system at sites of high enzyme activity is avoided, with consequent avoidance of diffusion of free naphthol prior to coupling. The dye produced is of a highly contrasting color and its insolubility in ethanol and xylol enables the mounting of tissues in synthetic media. The procedure is applicable to both Formalin and acetone fixed tissues, either frozen sections or paraffin ribbons, adequate pigment deposition being achieved after incubation periods of 15-120 minutes.