Quantitative Bone Histomorphometry Analysis

Cross-sections (50 μm thickness) of periosteal regions of undecalcified diaphysis tibial bone of each mouse were obtained using an Isomet-Slow Speed Bone Cutter (Buehler, Lake Bluff, IL, USA). Images were captured using Leica-Qwin software (Leica Microsystems Inc., Buffalo Grove, IL, USA) and bone forming rate/bone surface (BFR/BS) and MAR were calculated. Tetracycline was injected 30 (20 mg/kg) days before termination of treatment, whereas calcien (20 mg/kg) 24 h before termination of treatment.^{36 (link)} For TRAP histochemistry, femur bones were fixed in 4% formaldehyde and decalcified using EDTA solution. The bones were dehydrated, sectioned in 5 μm thick size, deparaffined and TRAP staining was performed according to previously published protocol. Histological section of bone stained for TRAP activity was analyzed with bioquant software. Analysis of osteoclast number and osteoclast surface was performed according to standardized protocols of the American Society for Bone and Mineral.^{38 (link)}