Ultrastructural cytochemistry and immunocytochemistry of sulfated glycosaminoglycans in epiphyseal cartilage.

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Abstract
Cartilage proteoglycan monomers contain sulfated glycosaminoglycans (GAG), which consist of chondroitin 4-and 6-sulfate, and keratan sulfate. Newborn rat epiphyseal cartilage was examined to demonstrate the nature of reactive sites in the matrix granule with the high Fe diamine (HID) and HID-thiocarbohydrazide-Ag proteinate (HID-TCH-SP) methods for sulfated glycoconjugates and an immunoferritin method specific for chondroitin 4-sulfate. HID strongly stained matrix granules in osmicated specimens but weakly stained these granules in unosmicated specimens. HID-TCH-SP staining produced 2 types of stain deposits, averaging 11 and 7 nm in diameter, in the matrix granule and was not influenced by postosmication. Testicular hyaluronidase digestion of specimens removed most of the 11 nm stain deposits in the matrix granule. The size of the HID-TCH-SP reaction products appeared to be related to MW and/or sulfate content of the GAG chains, as evidenced by the presence of large stain precipitates localizing hyaluronidase digestable chondroitin 4-sulfate and 6-sulfate and small stain precipitates over hyaluronidase resistant material presumed to be keratan sulfate. Immunostaining of chondroitinase-treated specimens with rabbit antibody specific for the unsaturated uronic acid residue linked to N-acetylgalactosamine-4-sulfate followed by ferritin-conjugated goat anti-rabbit IgG localized chondroitin sulfate in a distribution similar to that observed for HID-TCH-SP stain deposits in matrix granules. Thus both HID-TCH-SP and immunostaining methods allow differential ultrastructural localization of chondroitin sulfate in cartilage tissue and identify the matrix granules as the proteoglycan monomer(s).