Ultrastructural location of complex carbohydrates in developing rat incisor enamel

The ultrastructural distribution of complex carbohydrates in an early formation stage of rat incisor enamel was investigated by staining with the periodic acid-thiocarbohydrazide-silver proteinate reaction (PA-TCH-SP) for vicinal glycol-containing glycoconjugates, the phosphotungstic acid-chromic acid mixture (PTA) for glycoproteins, and the cationic dyes alcian blue or bismuth nitrate for sulfated glycoconjugates. In order to remove selectively sulfated complex carbohydrates, half of the serial sections obtained were digested with a bovine testicular hyaluronidase prior to staining. Far fewer electron-dense deposits were observed with the PA-TCH-SP method on hyaluronidase-treated sections, especially those subsequently treated for 48 hours with TCH. On the other hand, the minimal staining obtained with PTA was much more intense on sections treated with hyaluronidase where linear fiberlike structures were observed. With cationic dyes, staining of dotlike alignment structures and ground substance was obtained but was completely abolished by hyaluronidase treatment. Cuprolinic blue in a critical electrolyte concentration, ruthenium hexamine trichloride used with aldehyde during fixation, as well as rapid-freezing followed by freeze-substitution validate that this dotlike distribution is not an artefact of processing. The staining results demonstrated that the glycoproteins and sulfated complex carbohydrates in developing rat incisor enamel each display a specific distribution pattern. The glycoproteins were present as fiberlike structures and the sulfated carbohydrates appeared as dotlike formations located close to the surface of the fiberlike structures, and/or in the spaces between them.