THE USE OF PHENAZINE METHOSULFATE FOR IMPROVING THE HISTOCHEMICAL LOCALIZATION OF KETOSE REDUCTASE (l-IDITOL: NAD OXIDOREDUCTASE, OR SORBITOL DEHYDROGENASE)

Abstract

An improved histochemical localization of the activity of ketose reductase, a nicotinamide adenine dinucleotide-dependent enzyme, was achieved by using an incubating medium containing phenazine methosulfate. Fixed sections of mouse male accessory reproductive glands, liver and kidney reacted faster in the presence of phenazine methosulfate, yet without the false localizations found with media lacking this compound. At the cytologic level, phenazine methosulfate caused a fine deposition of formazan in the cytoplasm of all reactive tissues, whereas without it granules presumed to represent mitochondria were stained. Phenazine methosulfate is believed to produce these effects by serving as an intermediate electron acceptor between reduced coenzyme and tetrazolium. Thus detectable diffusion of reduced coenzyme did not occur because phenazine methosulfate improved trapping. The concentrations of both phenazine methosulfate and coenzyme were critical in obtaining accurate cytologic localization. The use of cyanide with phenazine methosulfate, although necessary with unfixed tissues, was not essential with fixed tissues.