Diazotized acetoxymercuric aniline in a postcoupling procedure for β-glucuronidase and acid phosphatase using naphthol AS-BI β-d-glucosiduronic acid and naphthol AS-BI-phosphoric acid respectively as substrates has yielded excellent 2-µ sections from Araldite-embedded specimens for light microscopy and correlating electron micrographs for both enzymes. The reaction of the red pigment product with thiocarbohydrazide is believed to establish a mercury-sulfur linkage which is apparently responsible for the pigment's surviving ethanol dehydration and epoxy embedding. Osmication of the tissue before dehydration appears to increase electron density of the product apparently by either chelation to the pigment product, by reduction of osmium tetroxide or by its decomposition with mercuric sulfide. The two acid hydrolases have been demonstrated within the cisternae of the Golgi and the endoplasmic reticulum and within the nuclear envelope of parenchymal cells of mouse and rat liver. Intracytoplasmic lipid inclusions in the livers of both C57 and C3H starved mice have shown a deposition of reaction product for acid phosphatase, whereas only C57 starved mice have shown a deposition of reaction product for β-glucuronidase. Lipid droplets in the rat preputial gland were sites high for β-glucuronidase, as has previously been demonstrated by two dissimilar techniques. Particularly helpful in evaluating the results of electron microscopy has been the use of physiologic controls as, for example, by comparison of findings made with the "high" tissue β-glucuronidase strain, C57, versus those of the "low" tissue β-glucuronidase strain, C3H. Localization of electron-dense product in lysosomes was clear, but not all of this can be attributed unequivocally to enzyme-produced density. The limitations of the technique and the question of nonspecific uptake of diazotized mercuric aniline are discussed. The findings for both hydrolases support the concept of the dual localization of acid hydrolases in lysosomes and in endoplasmic reticulum.
