Immunohistochemical observation of enamel protein localization in secretory ameloblast by application of Brefeldin A in organ culture system of mouse molar tooth germs

Abstract

Immunolocalization of enamelprotein in secretory ameloblasts (SA) before and after treatment with Brefeldin A (BFA) was done in 12 day-cultured mouse molar tooth germs using a polyclonal antibody which cross reacted with both amelogenin and enamelin.
Tooth germs from 16.5-day-old mouse embryos (vaginal plague day 0) were cultured in an organ culture system using BGJb culture medium, supplemented with 10% FCS, 2 mM Glutamine, 2 mM Glysine and 0.5 mM Vitamin C. In the experimental group, on culture day 12, BFA was introduced to whole tooth germs in the culture medium for different periods of time. For the control group, tooth germs were sampled at time zero, before treatment with BFA. The tooth germs treated with and without BFA were fixed for ultrastructural and immunohistochemical observations.
BFA treatment caused vesiculation of the Golgi lamellae following the disassembly of the Golgi complex, distention of the cisternae of the rER, in which accumulation of fine granular material was observed, and the disappearance of secretory granules in the Tomes' processes. Immunolabeling of the fine granular material which was observed in the distended cisternae of the rER, in the secretory ameloblasts treated with BFA, proves that the accumulated material is enamel protein, therefore confirming that enamel protein is processed in the rER.
In BFA untreated secretory ameloblasts, labeling was observed in the enamel matrix, Golgi complex, and secretory vesicles. No labeling was observed in the rER.