Histologic, immunohistochemical, and histomorphometric analyses.
All analyses were performed essentially as described previously [62,63]. Briefly, embryonic mice were removed from timed pregnant dams at E14.5 and E16.5 and fixed intact for 36 h in 4% paraformaldehyde, rinsed thoroughly in PBS, and processed for paraffin embedding. Serial 4-μm sections were cut on a modified Leica RM 2155 rotary microtome (Leica Microsystems, Richmond Hill, Ontario, Canada), stained with a 1:600 dilution of the AD1 anti-Sam68 antibody [46] and counterstained with either methyl green or hematoxylin.
Adult mice were given an intraperitoneal injection of 30 mg/kg calcein at 7 days and 30 mg/kg tetracycline at 2 days prior to sacrifice to label actively mineralizing surfaces. After overnight fixation in 4% paraformaldehyde and rinsing in PBS, the left femur and tibia were embedded in polymethylmethacrylate (MMA) or a mixture of 50% MMA and 50% glycolmethacrylate (GMA). Serial 4- to 6-μm sections of MMA-embedded tissues were left unstained or stained with von Kossa and toluidine blue or with toluidine blue alone, while 4-μm MMA-GMA sections were stained for TRAP and ALP activity. Images were captured using a Leica DMR microscope (Leica Microsystems) equipped with a Retiga 1300 camera (Qimaging, Burnaby, British Columbia, Canada) and the primary histomorphometric data obtained using Bioquant Nova Prime image analysis software (Bioquant Image Analysis Corp, Nashville, Tennessee, United States). Nomenclature and abbreviations conform to those recommended by the American Society for Bone and Mineral Research [50].