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Journal of Histochemistry and Cytochemistry, Vol. 45, 1189-1196, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Pre-osteoblastic Proliferation Assessed with BrdU in Undecalcified, Epon-embedded Adult Rat Trabecular Bone

Odile Baroua, Norbert Larochea, Sabine Pallea, Christian Alexandrea, and Marie-Hélène Lafage-Prousta
a Laboratoire de Biologie du Tissu Osseux, University of Saint-Etienne, Saint-Etienne, France

Correspondence to: Marie-Hélène Lafage-Proust, Laboratoire de Biologie du Tissu Osseux, Faculté de Médecine, 15 Rue A. Paré, 42023 Saint-Etienne Cedex 2, France.

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry in undecalcified adult rat tibiae to study cell kinetics in various bone compartments: primary and secondary spongiosae, periosteum, and bone marrow. Several regimens of BrdU administration were tested (IP injections and osmotic minipumps). We compared LR White resin, methylmethacrylate, and Epon-araldite embedding, microwave irradiation for antigen retrieval, several concentrations of sodium ethoxide for deplastification, and various DNA denaturation procedures. Paraffin-embedded decalcified tibiae and Epon-embedded bowel were used as positive controls. The best results were obtained in rats labeled with 40 mg of BrdU for 72 hr using osmotic minipumps. The procedure using a Microprobe system in Epon-embedded bone tissue with a sodium ethoxide concentration of 50% for two intervals of 20 min provided the best staining quality and tissue preservation. Labeled pre-osteoblastic cells and bone marrow cells could be counted. Epon embedding allowed preservation of tetracycline double labeling performed 1 to 5 days before sacrifice. The number of labeled pre-osteoblastic cells was correlated with the double-labeled surface area measured histomorphometrically. (J Histochem Cytochem 45:1189-1195, 1997)

Key Words: bromodeoxyuridine, rat immunohistochemistry, plastic embedding, bone proliferation, osteoblast


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