Briefly, mice (P80) were injected (intraperitoneally) with fluorochrome-labeled calcein green (10 mg·kg−1, Sigma-Aldrich) twice (10-d interval). The mice were sacrificed 2 d after the second injection. The left tibias and femurs were fixed in 70% (vol/vol) ethanol overnight, embedded in methyl methacrylate, and sectioned at 7–10 μm thickness. Images were obtained using a Zeiss LSM 800 fluorescence microscope. The mineral apposition rate (MAR) in μm·d−1 and bone formation rate (BFR) [BFR = MAR × MS (mineral surface) / BS (bone surface)] were calculated by measuring double fluorescence at the trabecular bone and endocortical surfaces.
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