TRAP staining

Osteoclast formation was determined by detection of TRAP-positive cells as vendor’s instruction (Sigma-Aldrich). Briefly, cells were fixed for 30 s and incubated with a solution containing naphthol AS-BI phosphoric acid at 37 °C for 1 h. The naphthol AS-BI phosphoric acid hydrolyzed by TRAP coupled immediately with fast garnet GBC to form insoluble maroon dye deposited at sites of activity. Stained cells were subjected to a counterstain with a hematoxylin solution. Osteoclasts were determined as TRAP-positive, multinucleated giant cells using light microscopy. The number of TRAP-positive cells and the number of nuclei per TRAP-positive cell in each well were counted, and the morphological features of osteoclasts were also photographed.