High-resolution immunocytochemistry of noncollagenous matrix proteins in rat mandibles processed with microwave irradiation

The mineral phase in calcified tissues represents an additional factor to b e considered during their preservation for ultrastructural analyses. Microw ave (MW) irradiation has been shown to facilitate fixative penetration and to improve structural preservation and immunolabeling in a variety of soft tissues. The aim of the present study was to determine whether MW processin g could offer similar advantages for hard tissues. Rat hemimandibles were i mmersed in 4% formaldehyde + 0.1% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.2, and exposed to MWs for three periods of 5 min at tempe ratures not exceeding 37C. They were then decalcified in 4.13% EDTA, pH 7.2 , for 15 hr, also under MW irradiation. Osmicated and non-osmicated samples were dehydrated in graded concentrations of ethanol and embedded in LR Whi te resin. Sections of incisor, molars, and alveolar bone were processed for postembedding colloidal gold immunolabeling using antibodies against amelo blastin, amelogenin, bone sialoprotein, or osteopontin. Ultrastructural pre servation of tissues was in most cases comparable to that obtained by perfu sion-fixation, and there was no difference in distribution of labeling with those previously reported for the antibodies used. However, the immunoreac tivities obtained were generally more intense, particularly at early stages of tooth formation. Amelogenin was abundant between differentiating amelob lasts and labeling for osteopontin appeared over the Golgi apparatus of odo ntoblasts after initiation of dentine mineralization. We conclude that MW i rradiation represents a simple method that can accelerate the processing of calcified tissues while yielding good structural preservation and antigen retention.