The demonstration of acid phosphatase in vitro cultured tissue cells. Studies on the significance of fixation, tonicity and permeability.

Abstract

1. Methods for the fine structural demonstration of acid phosphatase were studied in monolayers ofin vitro cultured cells after fixation with glutaraldehyde. 2. Inactivation of enzyme activity occurred rapidly during the initial phase of glutaraldehyde fixation. 3. Fixation for more than 5 min did not cause further marked inactivation of enzyme activity. 4. Stabilization of the cells for cytochemical incubations required a fixation for at least 30 min in glutaraldehyde. 5. The total osmolality of the fixative was of minor importance, in contrast to the major importance of effective osmolality, for obtaining optimum cytochemical and ultrastructural results. 6. Following proper fixation, the osmotic strength of the washing and incubation solutions was not critical. 7. With short fixation times, the composition of the washing and incubation solutions was of major importance. 8. Dimethyl sulphoxide in washing and incubation media was effective in shortening incubation times (thereby preventing the occurrence of unspecific precipitates and derangement of fine structure).