PRE-OSTEOBLASTIC PROLIFERATION ASSESSED WITH BRDU IN UNDECALCIFIED, EPON-EMBEDDED ADULT-RAT TRABECULAR BONE

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry in undecalc ified adult rat tibiae to study cell kinetics in various bone compartm ents: primary and secondary spongiosae, periosteum, and bone marrow. S everal regimens of BrdU administration were tested (IF injections and osmotic minipumps). We compared LR White resin, methylmethacrylate, an d Epon-araldite embedding, microwave irradiation for antigen retrieval , several concentrations of sodium ethoxide for deplastification, and various DNA denaturation procedures. Paraffin-embedded decalcified tib iae and Epon-embedded bowel were used as positive controls. The best r esults were obtained in rats labeled with 40 mg of BrdU for 72 hr usin g osmotic minipumps. The procedure using a Microprobe system in Epon-e mbedded bone tissue with a sodium ethoxide concentration of 50% for tw o intervals of 20 min provided the best staining quality and tissue pr eservation. Labeled pre-osteoblastic cells and bone marrow cells could be counted. Epon embedding allowed preservation of tetracycline doubl e labeling performed 1 to 5 days before sacrifice. The number of label ed pre-osteoblastic cells was correlated with the double-labeled surfa ce area measured histomorphometrically.