Electron microscopical enzyme histochemistry on unfixed tissues and cells. Bridging the gap between LM and EM enzyme histochemistry

Summary nciple, enzyme histochemistry should be performed on unfixed tissues and cells to avoid inhibition of enzyme activity by chemical fixation. For EM enzyme histochemistry, unfixed tissue specimens include fresh tissue blocks, non-frozen tissue chopper sections, cryostat sections and cell preparations. Studies on localization of enzyme activity at the ultrastructural level in unfixed specimens, be it fresh or frozen, are reviewed here. Preservation of ultrastructural morphology is discussed with special attention to the effects of freezing. It is concluded that unfixed cryostat sections are the best alternative for EM histochemistry of tissues, when interposing a semipermeable membrane in between cryostat section and gelled incubation medium. It is an adequate method to preserve structural integrity of unfixed tissue on the one hand and to avoid inactivation of the enzyme by chemical fixation on the other. For EM cytochemistry on individual cells, a better preservation of ultrastructure may be obtained because freezing can be avoided, but mild pretreatment with a fixative or detergent may be necessary to permeabilize cellular membranes for demonstration of intracellular enzyme activity.