Histochemical staining of cells containing flavoenzyme D-amino acid oxidase based on its enzymatic activity: Application of a coupled peroxidation method.

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Abstract
A histochemical method for the demonstration of D-amino acid oxidase activity in rat kidney, liver and brain is described. The method is a peroxidase-coupled procedure, which is based on the intensifying effect of nickel ions on 3,3''-diaminobenzidine-based product formation with peroxidase. The substrates used were D-proline and D-alanine, of which the former showed more intense staining. The histochemical reaction was suppressed by benzoate, a competitive inhibitor. There was no staining on omission of a substrate or peroxidase from the incubation medium. The method was specific for D-amino acid oxidase. The oxidase in kidney and brain showed resistance to the fixatives used (e.g. 8% glutaraldehyde), while the enzyme in hepatocytes did not. The reaction product was black, electron dense, stable and strictly confined to cells containing the oxidase. The method was successfully applied to the histochemical identification of the type of cell containing the oxidase at both the levels of light and electron microscopy. The oxidase activity was shown to be present in hepatocytes in the liver, proximal tubule cells in the kidney, and only astrocytes, including Bergmann glial cells, in the brain. In the midbrain, the oxidase was almost exclusively restricted to the tegmentum.