HISTOCHEMICAL LOCALIZATION OF SPECIFIC OXIDATIVE ENZYMES III. EVALUATION STUDIES OF TETRAZOLIUM STAINING METHODS FOR DIPHOSPHOPYRIDINE NUCLEOTIDE DIAPHORASE, TRIPHOSPHOPYRIDINE NUCLEOTIDE DIAPHORASE AND THE SUCCINDEHYDROGENASE SYSTEM

EMMANUEL FARBER ¹, WILLIAM H. STERNBERG ¹, CHARLES E. DUNLAP ¹, and CONCETTA M. LOUVIERE ¹

¹ Departments of Pathology and Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana

Histochemical stains are methods for the visual demonstration of the biochemical properties of organized tissues. They differ from "ordinary" tissue stains only in so far as they lend themselves to chemical interpretation and are reliable only to the extent that they accurately reflect the presence and localization of specific chemical substrates or activities. In this paper we have described the results of a number of experiments designed to test the reliability and usefulness of tetrazolium salts as histochemical indicators of specific oxidative enzyme activities.

Control studies dealing with substrate specificity and inhibitors gave further evidence of the specificity of tetrazolium staining methods for the succinic dehydrogenase system.

Studies on the possibility of diffusion and nonspecific deposition of reduced blue tetrazolium and neotetrazolium indicated that the blue reduction products (diformazans) of each do not deposit nonspecifically from the medium and once deposited are firmly fixed in the tissues. The red reduction products (monoformazans) may deposit nonspecifically from the medium. Hence their deposition and localization must be interpreted with caution. The red monoformazans of these two salts can readily be converted to blue diformazans in tissue sections by non-enzymatic reducing agents.

Blue tetrazolium in the unreduced form is reversibly bound in the cytoplasm of all cells of the rat kidney but not in the nuclei. In the staining process it is probable that tetrazolium, bound in the cytoplasm, is reduced to formazan irrespective of the continued presence of free tetrazolium in the medium. Since no binding of tetrazolium is demonstrable in any of the cell nuclei, the failure of nuclei to stain does not necessarily indicate the absence of enzyme. It was found that two other hydrogen acceptors, methylene blue and pyocyanin, are also bound by the cytoplasm of cells and when either of these compounds is added to tetrazolium staining media, tissue staining is greatly accelerated and enhanced.

Relative enzyme activities determined quantitatively in tissue taken from different anatomical regions of the rat kidney correspond fairly well with the intensity of histochemical staining.

Tetrazolium methods are now available for demonstrating the succinic dehydrogenase system, diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. These methods appear to satisfy most of the criteria which we have adopted for the specificity and reliability of histochemical stains. They yield preparations which are quite satisfactory for histochemical purposes. However, it has not as yet been demonstrated that the formazan deposits accurately indicate the intracellular localization of enzyme activity and hence cytochemical interpretations are open to question.

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