Optical microscope

The liver tissue samples were embedded in paraffin, sectioned into 4-μm slices, and stained with hematoxylin and eosin (H&E). Oil red O staining was performed using frozen liver tissue sections, LO2 cells, and LO2 stable cell lines of FST overexpression or knockdown. The cells were treated as previously described. The stained area in 10 random fields was quantified using an optical microscope (Zeiss, Germany) and Image-Pro Plus 6.0 software (Media Cybernetics, USA).

Paraffin-embedded liver sections were deparaffinized, hydrated, thermally repaired with 0.01 M sodium citrate buffer, and then incubated in 3% hydrogen peroxide for 10 min to eliminate endogenous peroxidase activity. Next, the sections were incubated with 5% BSA for 1 h at 37°C and primary antibody against FST (#60060-1-Ig, Proteintech, China) at 4°C overnight and then washed with phosphate-buffered saline three times.
For IHC staining, the sections were incubated with secondary antibody and StreptAvidin—Biotin Complex (SABC) for 30 min each (#SA1050, BOSTER, China), and then 3.3’-diaminobenzidine (DAB; #AR1022, BOSTER, China) was added for 3 min. Finally, hematoxylin staining was performed for cell counting. The IHC results were based on 10 random fields in each section using an optical microscope (Zeiss, Germany).
For IF staining, fluorescent secondary antibody (Alexa Fluor^® 488-labeled goat anti-mouse IgG(H+L); #ab150113, Abcam) was added for 1 h at room temperature, and then the cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min. The IF images were obtained using a confocal microscope (Zeiss, Germany).

Osteoclasts were fixed in 4% formaldehyde and osteoclasts differentiation was evaluated with a TRAP detection kit (Sigma-Aldrich, USA). Stained osteoclasts were observed with an optical microscope (ZEISS, Germany) and quantified by Image-J software. Total protein was extracted and the TRAP activity was measured by the TRAP assay kit (Beyotime Technology Inc, China).