Acid Hydrolases in Hela Cells: Comparison of Methods for Light Microscopy

TO distinguish lysosome populations of HeLa cells, acid phosphatase, /8-glu-curonidase, arylsulfatase and esterase were demonstrated using various substrates and couplers with different fixations, pHs and inhibitors. The substrates chosen were for acid phosphatase, naphthol AS-BI phosphate with fast red violet LB at pH 4.6; for β-glucuronidase, naphthol AS-BI β-D-glucuronide with fast red violet LB at pH 4.4; for arylsulfatase, p-nitrocatechol sulfate, with lead as the capturing ion, at pH 4.8 and 5.6; and for esterase, naphthol AS-D acetate with fast blue BB at pH 6.5. In the azo-dye methods, the coupling was always simultaneous and results were satisfactory with unfixed cells. For optimal demonstration of arylsulfatase, cells were fixed in glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, 2% for 24 hr or 6.25% for 2 hr, and washed for 1-9 days in 0.1 M veronal acetate buffer pH 7.2, 7.5% with respect to sucrose. Two groups of lysosomes were distinguished. One comprised small bodies, probably primary lysosomes, which lay in a cluster near the nucleus. They had quite stable membranes and were mostly acid phosphatase-positive. They sometimes contained β-glucuronidase or esterase, but rarely arylsulfatase. The other group included all the acid hydrolase-positive bodies scattered throughout the rest of the cytoplasm. They were mostly larger, with more labile membranes, and contained β-glucuronidase, esterase or arylsulfatase, but rarely acid phosphatase.