VARIATIONS IN FORMAZAN PRODUCTION IN MITOCHONDRIAL DEHYDROGENASE STUDIES ON OSMOTICALLY PROTECTED FRESH FROZEN SECTIONS

Abstract

The application of osmolar protection to mitochondria of cryostat sectioned material, and the subsequent effect produced upon the reduction of the two tetrazolium compounds nitro blue tetrazolium and iodonitrotetrazolium has been investigated. The incorporation of polyvinylpyrollidone at concentrations to provide mild hyperosmolarity in incubation media used to demonstrate intramitochondrial dehydrogenases, had the effect of producing a decrease in the amount of tetrazole reduced, and variations in the distribution pattern of the formazan of nitro blue tetrazolium. Variability in the distribution pattern was linked to the degree of thermal damage occasioned to the mitochondria during the preparations of the sections. This variability in result was not so pronounced when thermal damage was considerable. Omission of osmotic protection during incubation had the effect of producing a more uniform reduction of tetrazole. This study demonstrates the desirability of careful standardisation in the production of fresh frozen sections for dehydrogenase histochemistry, and it is recommended that osmolar protection should only be applied when the degree of thermal damage has been standardised. Failure to standardise will produce misleading results.