Inveon preclinical ct

To identify osteoclasts and osteoblasts, bones were fixed overnight in 4% paraformaldehyde (Sigma, St. Louis, MO, USA) in PBS at 4°C. The bones were decalcified in 10% EDTA for up to 5 weeks and embedded in paraffin, and then cut into 5-μm sections. Sections were stained with hematoxylin and eosin to visualize the osteoblasts. For osteoclast visualization, sections were stained with 225 μM naphthol AS-MX phosphate (Sigma), 0.84% N,N-dimethylformamide (Sigma), and 1.33 mM Fast Red Violet LB Salt (Sigma) in 50 mM sodium acetate (pH 5.0) containing 50 mM sodium tartrate and incubated for 30 min. After incubation, the sections were washed in distilled water and counterstained with 1% methyl green. We performed histomorphometric analysis using the Bioquant OSTEOII Program (Bioquant Image Analysis Corporation, Nashville, TN, USA). For μCT imaging, the femurs of mice were fixed and prepared for analysis. μCT imaging was performed on a μCT scanner (Inveon Preclinical CT, Siemens Healthcare, Hoffman Estates, IL, USA) at 40-μm slice thickness with an exposure time of 600 ms, photon energy of 70 keV, and current of 400 μA. The bone parameters (bone volume/total volume, bone surface area/bone volume, trabecular thickness, trabecular number, and trabecular spacing) were calculated from a 2.5 × 0.5 × 0.5 mm³ area of the distal femoral bone using the Siemens Inveon Software.