TRAP staining of cells was performed as previously described (20). Briefly, the cells were fixed in 4% PFA with 0.1 M cacodilic buffer, pH 7.2 (0.1 M sodium cacodilate, 0.0025% CaCl2) for 15 min at room temperature, extensively washed in the same buffer, and stained for TRAP according to the manufacturer's protocols. After washing with distilled water and drying, samples were observed under a Leica microscope (Leica Microsystems GmbH). Mature TRAP-positive multinucleated cells containing >3 nuclei were counted as osteoclasts in 10 randomly selected optical fields for each sample (magnification, ×20).

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