Histochemical and immunohistochemical stainings were performed as previously described 30 (link),31 (link). Briefly, dissected bones were fixed in 4% PFA for 48 h, decalcified in 0.5 M EDTA (pH = 7.4) for 4-5 days, and dehydrated in graded ethanol, before being embedded in paraffin. 5-μm-thick bone sections were stained with hematoxylin and eosin (H&E), osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) to quantify number and area of adipocytes, number of osteoblasts, and number of osteoclasts, respectively. Images were photographed using an Olympus CX31 microscope (Olympus, Hamburg, Germany). Primary antibody against OCN and the secondary antibody were purchased from Abcam (Cambridge, Britain). TRAP Staining Kit was obtained from Sigma (St. Louis, USA).
Histomorphometric analysis was conducted to test dynamic bone formation. Briefly, the mice were intraperitoneally injected with 10 mg/kg body weight calcein (Sigma) dissolved in PBS at 10 and 3 days before sacrifice. The collected femurs were fixed in 4% PFA for 48 h, dehydrated in increasing concentrations of ethanol and sectioned without decalcification (60-μm sections). calcein double labeling was observed by a fluorescence microscope (Leica). Mineral apposition rates (MAR) of trabecular bone were determined using Image-Pro Plus 6 software.
Histomorphometric analysis was conducted to test dynamic bone formation. Briefly, the mice were intraperitoneally injected with 10 mg/kg body weight calcein (Sigma) dissolved in PBS at 10 and 3 days before sacrifice. The collected femurs were fixed in 4% PFA for 48 h, dehydrated in increasing concentrations of ethanol and sectioned without decalcification (60-μm sections). calcein double labeling was observed by a fluorescence microscope (Leica). Mineral apposition rates (MAR) of trabecular bone were determined using Image-Pro Plus 6 software.
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