RANKL-Induced Osteoclastogenesis Imaging

We seeded 5 × 10⁴ RAW 264.7 cells/mL in a confocal chamber slide and pre-treated them with 10 μg/mL IPA for 2 h. Subsequently, we treated the cells with 100 ng/mL RANKL in the presence of IPA for 9 h and 5 min. IF staining was performed as described by Cho et al. with some modification [35 (link)]. The cells were incubated overnight at 4 °C with the following primary antibodies: anti-NFATc1 (1:100), anti-c-Fos (1:100), and anti-p65 (1:100). Subsequently, the cells were incubated for 1.5 h with the following secondary antibodies at room temperature: Alexa fluor488-labeled goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody and Alexa fluor488-labeled goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody (1:800; Thermo Fisher Scientific, Inc., Foster City, CA, USA). Nuclei were stained with 40 μg/mL Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Eventually, fluorescent signals were detected using an LSM 700 Zeiss confocal laser scanning microscope (Carl Zeiss, Jena, Germany).