Bone Histomorphometry: Calcein Labeling

For bone histomorphometric analysis on bone formation rates, double-time calcein labeling was performed according to previous studies 53 (link), 55 (link). At 16 days and 2 days prior to sacrifice, mice received intraperitoneal injections of 20 mg/kg calcein (Sigma-Aldrich, USA) dissolved at 2 mg/mL in PBS supplemented with 1 mg/mL NaHCO₃ (Sigma-Aldrich, USA). calcein was injected at 10 μL/g each time away from light via the right lower quadrant of the abdominal area with mice in a head-down position. At sacrifice, the right femora were isolated, fixed in 80% ethanol, embedded in methyl methacrylate, and sagittally sectioned into 30 μm slices using a hard tissue slicing machine (SP1600; Leica, Germany) away from light. Cortical endosteum surfaces were evaluated by a fluorescence microscope (STP6000; Leica, Germany) with an excitation wavelength of 488 nm. Quantification was performed using the parameters of mineral apposition rate (MAR) and bone formation rate (BFR) by the ImageJ 1.47 software and the related calculation 56 (link).