Immunofluorescence Staining of RANKL Protein

Immunofluorescence staining was performed as previously described (19 (link)). Briefly, cell monolayers were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.01% Triton X-100 in PBS at room temperature for 10 min. After blocking with 3% BSA at room temperature for 1 h, cells were incubated with a primary anti-RANKL mouse monoclonal antibody (1:50; cat. no. ab45039; Abcam) at 4°C overnight, followed by incubation with an Alexa Fluor 555 donkey anti-mouse secondary antibody (1:500; cat. no. ab150106; Abcam) in the dark at room temperature for 1 h. The coverslips were mounted using DAPI solution (Invitrogen; Thermo Fisher Scientific, Inc.). Slides were viewed using a fluorescent microscope (Imager Z1; Zeiss AG). The fluorescence intensity was analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).