Bromodeoxyuridine immunohistochemistry of epon-embedded undecalcified bone in a canine fracture healing model

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry of paraffin-, methyl methacrylate (MMA)-, and epon-araldite (epon)-embedded canine bone specimens to establish an optimal technique for studying cell kinetics of fracture healing in a canine tibial gap model. Dogs were sacrificed 4 months after tibial ostectomy and 1 hr after i.v. injection of BrdU (100 mg/kg). BrdU immunohistochemical staining with a peroxidase-labeled streptavidin-biotin (LAB-SA) method was performed on thin sections of tibia fixed in 70% ethanol and embedded in paraffin, MMA, or epon. Thin section of small intestines fixed in 70% ethanol and 10% neutral buffered formalin and embedded in paraffin, MMA, or epon were BrdU-stained and served as a model for proliferating tissue. Good and consistent BrdU immunostaining without detachment of bone sections was obtained for epon-embedded undecalcified bone sections. BrdU-positive cells were easily identifiable, in contrast to negligible background staining. BrdU-labeled osteoprogenitor cells and osteoblasts were observed around and on the surface of woven bone in external and internal callus of the ostectomy gap. Nuclei of osteocytes were not labeled. In contrast to the epon-embedded specimens, BrdU immunostaining of paraffin-embedded decalcified and MMA-embedded undecalcified bone specimens was unsatisfactory. The results of this study suggest that BrdU immunohistochemistry of ethanol-fixed, epon-embedded, undecalcified canine bone sections is a technique suitable for study of fracture healing with the described methodology.