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Microwave Irradiation of Ethanol-fixed Bone Improves Preservation, Reduces Processing Time, and Allows Both Light and Electron Microscopy on the Same Sample

Olivier Laboux¹, Natalie Dion², Victor Arana-Chavez, Louis-Georges Ste-Marie and Antonio Nanci

Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Québec, Canada (OL,VA-C,AN), and Metabolic Bone Diseases Laboratory, Centre de Recherche du CHUM, Hôpital Saint-Luc, Montréal, Québec, Canada (ND, L-GS-M)

Correspondence to: Dr Antonio Nanci, Faculty of Dentistry, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, QC, Canada H3C 3J7. E-mail: antonio.nanci{at}umontreal.ca

Methylmethacrylate (MMA) embedding is routinely used for histomorphometry of undecalcified bone preserved by prolonged immersion in ethanol, a procedure that yields poor ultrastructural detail. Because microwave irradiation (MWI) facilitates penetration of fixatives, we have investigated whether it can improve preservation by ethanol. Rat tibiae, some labeled with tetracycline, and a human iliac crest biopsy were immersed in 70% ethanol and dehydrated, both under MWI, for a total processing time of 7 hr. Controls were not irradiated, and all specimens were embedded in MMA at 4C. They were then processed for histomorphometry, histochemistry, structural analysis, and immunolabeling. The results showed that histological preservation was improved with MWI. Static bone formation and resorption parameters and rate of mineral apposition were similar to those of conventionally processed specimens. Mineral distribution, as visualized by von Kossa staining and backscattered electron imaging, was not affected. Alkaline phosphatase and tartrate-resistant acid phosphatase activity, as well as immunolocalization of bone sialoprotein and osteopontin, were readily visualized. Ultrastructurally, osteopontin exhibited a typical distribution in mineralization foci, between calcified collagen fibrils, and at cement lines. These data show that MWI improves preservation and permits application of a broad spectrum of analytical methodologies on the same bone sample while considerably reducing processing time. (J Histochem Cytochem 52:1267–1275, 2004)

Key Words: microwave irradiation • methylmethacrylate • bone • histomorphometry • histochemistry • immunolabeling • ultrastructure

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