Bone Histomorphometric Analysis Protocol

Bone histomorphometric analyses were performed on undecalcified long bones and vertebrae in accordance with previously described methods (71 (link)). Briefly, the bone was fixed with 4% paraformaldehyde, followed by dehydration in an ethanol series, and subsequently embedded in methyl methacrylate resin. The BV/TV ratio was measured by von Kossa staining. Osteoblast and osteoclast parameters were analyzed by toluidine blue staining (FUJIFILM Wako Pure Chemical 1B481) and TRAP staining (Cosmo Bio PMC-AK04F-COS), respectively. Analyses were performed using the OsteoMeasure analysis system (OsteoMetrics), according to standard protocols. Trabecular architecture was assessed in long bones using a μCT system (Comscan) at 90 kV and 45 μA, and the BV/TV ratio was measured using TRI/3D-BON software (RATOC) (25 (link)). Calcein was injected twice into mice with an interval of 3 days, and then, mice were sacrificed 2 days after the last injection.