Effects of a Triple Enzyme Digestion Method on a Diamine Reaction for Glycosaminoglycans of the Rat Aorta in Electron Microscopy

Abstract

Effects of a triple enzyme digestion method using glycosaminoglycans-degrading enzymes upon a diamine reaction have been tested for electron microscopic histochemical detection of glycosaminoglycans in extracellular matrix of the rat aorta. The triple enzyme digestion method consists of a sequence of chondroitinase B, testicular hyaluronidase and heparitinase. The results obtained by the present experimental and control studies indicated that dermatan sulfates, chondroitin sulfates (A and/or C) or heparan sulfates were apparently observed in various ultrastructural features of aortic extracellular matrix, such as bundle of collagen fibers and soluble matrix of interstitial space. Particularly, we found that both heparan sulfates and chondroitin sulfates (A and/or C) were detected in association with the basal lamina of smooth muscle cells and the external surface of elastic lamina, and in the latter heparan sulfates were frequently recognized as a mass, whereas chondroitin sulfates (A and/or C) were found intermittently along the external surface of elastic lamina. This suggests that the triple enzyme digestion method which combines the glycosaminoglycans-degrading enzymes with the diamine reaction can be postulated to represent efficient and useful technique for precise electron microscopic histochemical detection of the glycosaminoglycans in the extracellular matrix of the rat aorta.