A QUANTITATIVE APPRAISAL OF ENZYME HISTOCHEMICAL METHODS IN BRAIN TISSUE

The quantitative reliability of the histochemical methods for diphosphopyridine nucleotidediaphorase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, monoamine oxidase, and lactic dehydrogenase in brain tissue was tested by comparison with enzyme assays in tissue homogenates. The reaction for diphosphopyridine nucleotidediaphorase in formalin-fixed tissue can be considered a microchemical method; it adequately reflects the gradations among nuclei measured by assays in fresh tissue homogenates. The same was true for the reaction for succinic dehydrogenase in unfixed tissue sections. In general, however, enzyme histochemistry in unfixed sections was subject to substantial error due to leakage of enzyme activity into the incubation medium (or washing fluid) . This leakage was prevented by fixation of the tissue if fixation was compatible with the enzyme. The extent of leakage varied greatly among regions of a given section; neuropil generally leaked more than cell bodies. The rate of leakage also differed markedly among enzymes and was influenced by a variety of physical factors such as thickness of sections, storage, washing, etc. Thin cryostat sections were particularly susceptible to leakage. Working standards for the quantitative use of enzyme histochemical methods in brain tissue were developed. A combination of assay of homogenates with histochemistry is recommended, whereby the relative significance attributed to either method must be determined separately for every individual enzyme.