THE PROCEDURE FOR ULTRACYTOCHEMICAL DEMONSTRATION OF DEHYDROGENASES IN THE RAT ADRENAL CORTEX USING

This is a procedure for the ultracytochemical demonstration of 3ƒÀ-hydroxy-steroid dehydrogenase (3ƒÀ-HSD) and glucose-6-phosphate dehydrogenase (G6PD) and the localization of these enzymes in the adrenocortical cell of rat is presented. The procedure involves pre-fixation of tissues, tissue sectioning and incubation of specimens. Brief fixation (for 30 min) in a mixture of glutaraldehyde and formaldehyde (0.25 %:1 % and 2 % : 2%) or 2.7 % glutar-aldehyde was excellent to preserve both the activity of 3ƒÀ-HSD and G6PD and fine cellular structure. Unfrozen sections obtained by Vibratome (Oxford) were superior to frozen sections obtained by a cryostat for preservation of the ultrastructure and enzyme activity of the cell. Sections used were 40-100 ƒÊm in thickness. In the present method dehydroepiandrosterone (DHA), etio-cholane-3ƒÀ-17-one (etiocholane) and pregnenolone were utilized as substrate, potassium ferricyanide as a final electron acceptor instead of tetrazolium salt, and phenazine methosulfate (PMS) as an intermediate electron carrier. As a result, the activity of 3ƒÀ-HSD was localized in the cytoplasmic matri