Sc 18701

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).

Tissue biopsies from visceral adipose tissue, obtained during surgery, were stored at −80 °C until further processing. All subjects provided their written informed consent. All procedures that involved human samples were approved by the Ethics Committee of Bayi Clinical Medicine School of Nanjing Medical University. All relevant ethical regulations were followed. To examine MVP expression, paraffin sections were stained with an anti-MVP antibody (Santa Cruz, sc-18701, 1:50). To isolate SVFs, human adipose tissues were digested using collagenase type II (1.5 mg ml⁻¹, Sigma) at 37 °C for 40 min. After passing cells through a 200 μm cell strainer and centrifugation at 1000g for 10 min, the pellet containing the SVFs was then incubated with red blood cell lysis buffer. SVFs were resuspended in phosphate-buffered saline (PBS) supplemented with 1% fetal bovine serum (FBS, Gibco). CD14⁺ macrophages were purified using magnetic beads (BD Biosciences), according to the manufacturer’s instructions. Cells were immediately used for total RNA extraction.