4.6. Tartrate-Resistant Acid Phosphatase (TRAP) STAINING

The differentiated osteoclasts were fixed and stained for TRAP, an enzyme marker of osteoclasts, using a leukocyte acid phosphatase kit, according to the manufacturer’s instructions. Briefly, cells were stained with naphthol AS-BI phosphate, freshly diazotized fast gamer GBC, and tartrate solution for 1 h at 37 °C, followed by counter-staining with a hematoxylin solution. A TRAP-positive, multi-nucleated cell (>3 nuclei per cell) was counted as an osteoclast. The photos of stained osteoclasts were taken using a Nikon Ni-U upright fluorescence microscope equipped with MBF Stereo Investigator Software and a color camera. Image analysis was performed with ImageJ software (NIH Image, Bethesda, MD). The number of osteoclasts per well was calculated by averaging the counts from eight separated views.