Dynamic bone histomorphometry.

The left femur from each animal was dissected and fixed in 10% neutral buffered formalin. Samples were decalcified in ChelatorCal (BBC Biochemical) and embedded in paraffin, then cut into 4 μm slices. Femur sections were stained by either H&E or TRAP for osteoclast analysis. TRAP staining was carried out with the TRAP Stain Kit (TRAP/ALP Stain Kit, Wako Pure Chemical Industries Ltd.). Deparaffinized and rehydrated sections were incubated at 37°C for 30 minutes in TRAP substrate buffer, and then slides were mounted with VectaMount (Vector Laboratories). Images were visualized with an Olympus IX71/F22PH microscope and measurements performed with Bio-Quant software. The TRAP-positive osteoclast number (N. Oc) and surface (Oc.S) were normalized with BS.

To determine bone-formation rate (BFR) and mineralization, 6-week-old male mice were injected with 10 mg/kg calcein (MilliporeSigma) dissolved in 2.0% sodium bicarbonate (pH 7.0) 2 times a day for 7 days. Bones were fixed in 4% formalin. Femurs were embedded in paraffin blocks and sectioned to be 4 μm thick. Before histomorphometric analysis, femur metaphyseal sections were viewed with a fluorescent Nikon Digital Camera DXM1200 Microscope System, and 5 digital images per section were taken. The distance between the calcein lines (MAR) and the length of the calcein lines (mineralized surface) along the BS were measured to calculate BFR using ImageJ.