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Journal of Histochemistry and Cytochemistry, Vol. 49, 1099-1110, September 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

High-resolution Immunocytochemistry of Noncollagenous Matrix Proteins in Rat Mandibles Processed with Microwave Irradiation

Victor E. Arana–Chaveza,b and Antonio Nancib
a Laboratory of Mineralized Tissue Biology, Department of Histology and Embryology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil
b Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, University of Montreal, Montreal, Quebec, Canada

Correspondence to: Antonio Nanci, Département de Stomatologie, Faculté de Médecine Dentaire, Université de Montréal, CP 6128, Station Centre-Ville, Montréal, Québec, Canada H3C 3J7. E-mail: antonio.nanci@umontreal.ca

The mineral phase in calcified tissues represents an additional factor to be considered during their preservation for ultrastructural analyses. Microwave (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 processing could offer similar advantages for hard tissues. Rat hemimandibles were immersed 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 temperatures 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 White resin. Sections of incisor, molars, and alveolar bone were processed for postembedding colloidal gold immunolabeling using antibodies against ameloblastin, amelogenin, bone sialoprotein, or osteopontin. Ultrastructural preservation of tissues was in most cases comparable to that obtained by perfusion-fixation, and there was no difference in distribution of labeling with those previously reported for the antibodies used. However, the immunoreactivities obtained were generally more intense, particularly at early stages of tooth formation. Amelogenin was abundant between differentiating ameloblasts and labeling for osteopontin appeared over the Golgi apparatus of odontoblasts after initiation of dentine mineralization. We conclude that MW irradiation represents a simple method that can accelerate the processing of calcified tissues while yielding good structural preservation and antigen retention.

(J Histochem Cytochem 49:1099–1109, 2001)

Key Words: enamel proteins, noncollagenous matrix proteins, calcified tissues, extracellular matrix, microwave processing, immunocytochemistry


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