After euthanasia, mouse femurs were isolated and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. Next, specimens were decalcified in 10% EDTA, dehydrated, embedded in paraffin, and sectioned into 4.5 μm thick slices. Sections were permeabilized in PBS containing 1% Triton X-100, blocked in normal goat serum for 30 min at 37 °C and incubated with primary antibodies against MVP (1:100, sc-18701, Santa Cruz, Texas, America), CTSK (1:100, sc-48353, Santa Cruz) and Cleaved-Caspases3 (1:100, #9664, Cell Signaling Technology, Massachusetts, America) 4 °C overnight. On the second day, sections were incubated with secondary antibodies for 1 h at 37 °C and finally stained with 4’,6-diamidino-2- phenylindole (DAPI) (Beyotime, Shanghai, China) for 2 min at room temperature.
For in vitro studies, osteoclasts were harvested in 4% PFA for 15 min at room temperature, permeabilized with 0.5% Triton X-100 and blocked with normal goat serum at 37 °C for 1 h. Then, cells were co-stained with primary antibodies against MVP (1:100, sc-18701, Santa Cruz) and Fas (1:100, sc-74540, Santa Cruz) at 4 °C overnight. The next day, after washing with PBST, cells were incubated with secondary antibodies at 37 °C for 1 h. Nuclei were stained with DAPI. Images were captured using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
For in vitro studies, osteoclasts were harvested in 4% PFA for 15 min at room temperature, permeabilized with 0.5% Triton X-100 and blocked with normal goat serum at 37 °C for 1 h. Then, cells were co-stained with primary antibodies against MVP (1:100, sc-18701, Santa Cruz) and Fas (1:100, sc-74540, Santa Cruz) at 4 °C overnight. The next day, after washing with PBST, cells were incubated with secondary antibodies at 37 °C for 1 h. Nuclei were stained with DAPI. Images were captured using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).