Combined cytochemical and electron microscopic demonstration of beta-glucuronidase activity in rat liver with the use of a simultaneous coupling azo dye technique

Abstract

Rat liver was fixed in formal-cacodylate-sucrose and frozen sections were incubated in a simultaneous-coupling medium containing naphthol AS-BI glucuronide as substrate and hexazonium pararosanilin as the diazo reagent. By light microscopy, the sections demonstrated beta-glucuronidase activity as red discrete granules in the pericanalicular cytoplasm and as a generalized cytoplasmic stain in the parenchymal cells. Brief treatment of sections in cold ethanol prior to incubation markedly enhanced the staining for the enzyme and made it possible to demonstrate sufficient amounts of the reaction product in sections embedded in epoxy resin following dehydration and propylene oxide treatment. Electron microscopy revealed that the reaction product was moderately electron opaque and deposited in greater amounts in the vacuolated dense bodies and occasionally in the dense bodies which did not show obvious vacuoles. In each dense body, the deposits occurred preferentially at the edge as well as in the area surrounding the vacuoles in the matrix. Control sections incubated in the presence of glucosaccharo-1:4-lactone were devoid of the reaction product. No deposits of the reaction product were found in the nucleus, mitochondria, or microbodies. The limitations of the present cytochemical technique for use in electron microscopy are briefly discussed.