Immunocytochemical localization of enamelin proteins in developing bovine teeth

Enamelins were localized at both the light and electron microscopic level using an antienamelin monoclonal antibody and indirect immunogold methods. Bovine fetal incisors (crown-rump length 17-30 cm) were preserved in Karnovsky's fixative and embedded in Epon. For light microscopy, 2 microM thick sections were immunostained by the indirect method using the monoclonal antibody and goat anti-mouse IgG linked to 5 nM gold particles, followed by silver enhancement to increase the sensitivity of the method. For electron microscopy, thin sections were immunostained (indirect) with the antienamelin monoclonal antibody and goat anti-mouse IgG linked to 5 or 15 nM gold. Control samples were treated with an unrelated monoclonal antibody. The localization of enamelins was confined in the light microscopic sections to the extracellular enamel matrix. No gold staining was observed in the ameloblasts or other enamel organ cells even though the gold-silver technique is extremely sensitive. Ultrastructurally, enamelin was localized in the enamel extracellular matrix and associated ameloblasts. Both the crystal-containing and granular matrix were positively stained, with most gold particles being closely associated with the crystals. Counting of gold particles indicated more than 4 times as many amelogeninas enamelin-reactive antigenic sites in similar regions. Decalcification did not increase immunostaining with the anti-enamelin antibody in the extracellular matrix. Within ameloblasts, the gold particles were associated with secretory granules and Golgi complexes. Thus it appears that enamelins are synthesized in ameloblasts and secreted into the extracellular matrix in a similar manner to amelogenins and are preferentially associated with matrix hydroxyapatite crystals. Transient levels of enamelins within the ameloblasts are apparently too low to be detected by light microscopy.