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Localization and ultrastructure of proteoglycans during endochondral calcification or mineralization in the rat epiphyseal growth plate cartilage were investigated histochemically and by immunoelectron microscopy, using ruthenium hexammine trichloride and/or mouse monoclonal antibody (2-B-6) which recognizes chondroitin-4-sulphated glycosaminoglycans. The gold particles of the second antibodies were distributed solitarily, lineally or in small clusters in calcified cartilage as well as in the surrounding non-calcified matrix. The labelled matrix glycosaminoglycans in the longitudinal septa increased from the proliferative to the upper hypertrophic zones and remained constant in calcified cartilage. After fixation with ruthenium hexammine trichloride, matrix proteoglycans precipitated and were observed as a reticular network of strands. Immunogold-labelled antibodies for the demonstration of the glycosaminoglycans were localized on these strands. Proteoglycans in calcified cartilage were observed as a network of fine-filamentous structures after ruthenium hexammine trichloride, showing their non-precipitated native state. Immunostaining for chondroitin-4-sulphate indicated that there was no change in the concentration of glycosaminoglycans on the small calcified nodules in the areas where calcification was just beginning.

Histochemical and Immunoelectron Microscopic Localization of Proteoglycans in Calcified Cartilage of Rat Epiphyseal Growth Plate