Rvl 110 g

BMMs (7 × 10⁴ cells/well) were cultured in 48-well plates with α-MEM complete medium containing 30 ng/mL M-CSF, 100 ng/mL RANKL, and SOG (0–200 μM) for 4 d. Cells were fixed with 4% paraformaldehyde (PFA) for 20 min, and permeabilized with 0.5% Triton X-100 for 5 min. After washing three times with phosphate-buffered saline (PBS), phalloidin was used to stain F-actin for 30 min and DAPI was used to stain nuclei for 5 min. Fluorescent images were acquired on a fluorescence microscope (RVL-110-G; ECHO, San Diego, CA, USA).

Left femurs were fixed with 4% PFA for 24 h, and scanned by micro-CT using a Quantum GX microCT system (PerkinElmer, Waltham, MA, USA). The bone mineral density (BMD), bone volume/total volume (BV/TV), and trabecular number (Tb.N), trabecular thickness (Tb.th) and decreased trabecular separation/spacing (Tb.Sp) were measured.
Right femurs were demineralized in 10% ethylenediaminetetraacetic acid for 2 weeks. They were then embedded in paraffin, cut into 5 μm-thick sections, and stained with H&E and TRAP. Staining was visualized using a high-quality microscope (RVL-110-G; ECHO, San Diego, CA, USA).