A MODIFIED TECHNIQUE OF HISTOCHEMICAL DEMONSTRATION FOR N-ACETYL-β-HEXOSAMINIDASE

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A new histochemical method for β-hexosaminidase is presented as a modification of Hayashi's technique. N-acetyl-β-D-hexosaminides of Naphthol AS-BI were prepared by treating the anisidide with acetochlorohexosamines in acetone with an aqueous alkali. These compounds were recrystallized several times and used as the substrate. The pH of the medium could be desirably reduced near to the optimal pH. In simultaneous histochemical techniques, some diazonium salts were tested in order to determine their resistance to diffusion or their coupling rates. Fast red violet LB salt was more satisfactory as a coupling dye. No dye depositions were observed in the cells of various tissue samples above 50 μM. of N-acetyl-glucosaminolactone and N-acetyl-galactosaminolactone. Frozen sections of the tissues were incubated at 37℃ together with substrates in the presence of fast red violet LB salt at pH 4.4. Many fine granules associated with enzyme activity were found to be localized uniformly throughout the cytoplasm except for the nucleus. For the N-acetyl-β-glucosaminidase activity, the marked dye deposits were observed in some organs, such as epididymis and kidney. Dye deposits in the nerve cells of the brain and the spinal cord were also observed. For the N-acetyl-β-galactosaminidase activity, moderate dye deposits were found in rat tissue specimens. The latter enzyme, however, showed a tendency to be slightly less active than N-acetyl-β-glucosaminidase.