Use of glycine buffer for the lead citrate method of non-specific alkaline phosphatase cytochemistry.

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Abstract
To make reaction products finer in the enzyme cytochemical detection of nonspecific alkaline phosphatase (ALPase) by the lead citrate method, glycine-KOH buffer was tested for the incubation medium. Non-frozen sections of rat liver and kidney fixed with aldehyde fixatives were incubated in various media and observed by electron microscopy after the routine processing. With 250 mM of glycine, which is used for p-NPPase and H+,K+-ATPase cytochemistry, reaction products were coarse, but with 25 or 50 mM of glycine, they became comparable to or even finer than those of the original Mayahara method using Tris buffer (1967). Two different concentrations of the substrate, .beta.-glycerophosphate, 5 and 20 mM, did not make any difference in the results. Three different fixation conditions were tested, but the size of the reaction products remained the same with each incubation medium. Biochemical quantitation of liberated inorganic phosphate after incubating tissue homogenates with the cytochemical media did not show any drastic differences among them. The results indicate that the metal-chelating effect of glycine may affect the size of the lead phosphate deposits generated by ALPase.

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