Immunocytochemical examination of the presence of amelogenin during the root development of rat molars

The presence of enamel proteins, especially amelogenins, during root development has been a subject of controversy for a long time. Whereas some studies have reported the presence of enamel proteins on the root surface, others were not able to detect them at these places. Since microwave (MW) processing has been shown to improve the antigen retention in mineralised tissues, we have applied MW techniques to ultrastructurally analyse the presence of amelogenin during root formation in rat molars. Upper molar tooth germs from 12 and 13-day-old Wistar rats were fixed in 0.1% glutaraldehyde + 4% formaldehyde under MW irradiation. They were then decalcified in 4.13% EDTA, dehydrated in graded concentrations of ethanol and embedded in LR White Resin. Ultrathin sections were processed for post-embedding colloidal gold immunolabelling using a chicken egg yolk antibody against a 24 kDa rat amelogenin. Then, the grids were incubated with a rabbit anti-chicken IgG secondary antibody and with a protein A-gold complex. The immunoreactivity for 24-kDa amelogenin was detected in the cytoplasm of the epithelial diaphragm cells - the most apical portion of the Hertwig's epithelial root sheath (HERS), and in less amounts on the adjacent dental papilla extracellular matrix. Amelogenin was no longer observed at advanced stages of root dentinogenesis or later, during cementogenesis. The restricted presence of amelogenin at the early stages of root formation suggests that this protein may play a role in the differentiation of ectomesenchymal cells into root odontoblasts.