The procedure for ultracytochemical demonstration of dehydrogenases in the rat adrenal cortex using copper ferrocyanide.

Abstract

This is a procedure for the ultracytochemical demonstration of 3β-hydroxy-steroid dehydrogenase (3β-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 30min) in a mixture of glutaraldehyde and formaldehyde (0.25%: 1% and 2%: 2%) or 2.7% glutaraldehyde was excellent to preserve both the activity of 3β-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 40-100μm in thickness. In the present method dehydroepiandrosterone (DHA), etio-cholane-3β-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 3β-HSD was localized in the cytoplasmic matrix and intracristal space of mitochondria. The activity of G6PD was visualized mainly in the cytoplasmic matrix near the plasma membrane. The reaction did not take place in any cells incubated either in the substrate-free medium or in the medium markedly inhibited by respiratory chain inhibitors such as Rotenone and Antimycin A. The findings mentioned so far were evidently those of the specific ultracytochemical reaction. Several problems will be discussed in this paper concerning the procedure for the ultracytochemical demonstration.