Anti cox 4 antibody

White adipose tissue samples were fixed overnight in 4% paraformaldehyde, after which they were sliced into 5-μm-thick sections. Cells grown on coverslips were fixed in 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton X-100 (Sigma, United States). For histologic examination, tissue slices were stained with hematoxylin. For immunofluorescence and immunohistochemistry, tissue slices or cells were incubated with blocking buffer (1% bovine serum albumin) for 1 h, and then with anti-UCP1 (Proteintech, China), anti-COX IV antibody (Proteintech, China), or anti-MFN2 antibody (Cell Signaling Technology, United States) at 4°C overnight. After that, the tissue slices or cells were incubated with fluorescein isothiocyanate (FITC)–conjugated secondary antibody (for immunofluorescence) or horseradish peroxidase–conjugated secondary antibody (for immunohistochemistry) at room temperature for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Zhongshan Jinqiao, ZLI-9557). Immunofluorescence images were captured with an AV300-ASW confocal microscope (Olympus America Inc., Center Valley, PA, United States).

The following antibodies were used: anti-CYC1 antibody (Catalog number: 10242, Proteintech, China), anti-UQCRFS1 antibody (Catalog number: 18443, Proteintech, China), anti-UQCRC2 antibody (Catalog number: 14742, Proteintech, China), anti-UQCRC1 antibody (Catalog number: 21705, Proteintech, China), anti-UQCRB antibody (Catalog number: 10756, Proteintech, China), anti-NDUFS1 antibody (Catalog number: 12444, Proteintech, China), anti-SDHA antibody (Catalog number: 14865, Proteintech, China), anti-COXIV antibody (Catalog number: 11242, Proteintech, China), anti-OSCP antibody (Catalog number: 10994, Proteintech, China), anti-AMPK antibody (Catalog number: D63G4, Cell Signaling Technology), and anti-phospho-AMPKα (Thr172) antibody (Catalog number: 40H9, Cell Signaling Technology).