594 conjugated donkey anti rabbit antibody

The expression of p32 in myoblasts was examined by immunofluorescence analysis. Cells were seeded in a glass-bottom dish, fixed with ice-cold methanol for 20 min at room temperature, washed with PBS, and permeabilized for 10 min using 0.25% Triton X-100 (Sigma-Aldrich, St Louis, Missouri). Next, cells were blocked with an Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 60 min at room temperature on a rocking platform and then washed with PBS. The primary rabbit anti-p32 antibody (1:100 dilution, Proteintech, Chicago, IL, USA) was added to the cells and incubated overnight at 4 °C. Afterward, the cells were washed three times with PBS, after which the secondary antibody, the 594-conjugated donkey anti-rabbit antibody (1:200 dilution, Abcam, Boston, MA, USA), was added to the cells and incubated for 2 h at room temperature in the dark. Finally, the nuclei of the cells were stained with 4′,6-diamidino-2-phenylindole (Beyotime, Shanghai, China) for 10 min, and cell fluorescence was examined using a confocal laser scanning microscope (Zeiss LSM 710 META, Mannheim, Germany).

The frozen sections of subcutaneous fat stored at −20 °C until staining. The frozen sections prepared from the refrigerator were melted on a ventilated stage for 20 min. The frozen sections were fixed with 4% (v/v) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% (v/v) Triton X-100/PBS for 15 min. After treating with 3% (w/v) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% (w/v) BSA/PBS. After washing three times with PBS, the frozen sections were incubated with 594-conjugated donkey anti-rabbit antibody (1:200 dilution; Abcam, Cambridge, UK) in the dark for 2 h at room temperature. Subsequently, samples were stained with DAPI (Beyotime, Shanghai, China) for 10 min at room temperature. All samples were examined under a confocal laser scanning microscope (Zeiss LSM 710 META, Jena, Germany).