Light microscopical detection of D-amino acid oxidase activity in unfixed cryostat sections of rat kidney and liver using the cerium-DAB-cobalt-H2O2 procedure and a semipermeable membrane.

The two-step method for the light microscopical detection of oxidase activity using cerium ions and a visualization procedure with diaminobenzidine and nickel or cobalt ions as introduced by Anger-müller and Fahimi, has been improved. Inactivation by fixation and leakage of D-amino acid oxidase molecules were avoided by the application of unfixed cryostat sections adhered to a semipermeable membrane separating the sections from a gelled incubation medium. Optimum activity in rat kidney and liver was obtained by increasing the cerium concentration from 3 to 30 mM and the Tris-maleate concentration from 100 to 200 mM in the incubation medium for the first step. D-proline was used as substrate and a concentration of 20 mM gave maximum activity. The second visualization step was performed in a medium consisting of DAB, cobalt and hydrogen peroxide. Final reaction product was found in a granular form in the basal part of the epithelial cells of the proximal tubules and in liver parenchymal cells. The activity in periportal areas was much higher than in pericentral areas. Cytophotometric analysis revealed that a 30 times higher activity could be detected when using the membrane technique in comparison with incubations in aqueous media. The specificity of the reaction was proven by a nearly complete inhibition of the reaction by beta-hydroxybutyrate.