Osteoclasts cultured on TCPS and ECM were fixed with 4% paraformaldehyde for 15 min at room temperature. F-actin rings were stained with CytoPainter Phalloidin-iFluor 488 Reagent (Abcam) for 1 h according to the manufacturer’s instructions. Cell nuclei were counterstained with DAPI. Immunofluorescence images of F-actin rings were captured by an Olympus IX51 microscope. Using the ImageJ software (National Institutes of Health, Bethesda, MD, USA), at least 20 multinucleated osteoclasts in 10 randomly chosen fields were analyzed for the areas of F-actin rings. The number of nuclei within F-actin rings and total nuclei in all cells was quantified. The percent nuclei in F-actin was expressed as an index for fusion efficiency, which was calculated as nuclei within F-actin rings divided by total nuclei [25 (link)].
To investigate the role of NF-κB in osteoclastogenesis, osteoclasts were first fixed in ice-cold methanol for 15 min and then permeabilized for 5 min in 0.1% Triton X-100. The cells were blocked for 30 min with 1% BSA and incubated in appropriately diluted primary antibodies against p65 for 1 h. After a brief washing with PBS, an Alexa Fluor® 488 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific) was used. Cell nuclei were stained with DAPI for 5 min. Fluorescence images were obtained using an Olympus IX51 microscope.
To investigate the role of NF-κB in osteoclastogenesis, osteoclasts were first fixed in ice-cold methanol for 15 min and then permeabilized for 5 min in 0.1% Triton X-100. The cells were blocked for 30 min with 1% BSA and incubated in appropriately diluted primary antibodies against p65 for 1 h. After a brief washing with PBS, an Alexa Fluor® 488 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific) was used. Cell nuclei were stained with DAPI for 5 min. Fluorescence images were obtained using an Olympus IX51 microscope.