Av300 asw confocal microscope

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).

Cells grown on cover glasses were treated as designated. Cells were fixed in 4% paraformaldehyde and permeabilized by 0.5% Triton X-100. Following blocking with 3% bovine serum albumin, cells were serially incubated in rabbit anti-LC3B (Sigma-Aldrich) and Goat anti-rabbit Alexa Fluo488 (Invitrogen). Finally, cells were rinsed and mounted on cover glasses with Prolong Gold anti-fade reagent with 4′-6-diamidino-2-phenylindole (Invitrogen) and the immunostaining was observed under the AV300-ASW confocal microscope (Olympus America Inc., Center Valley, USA) with a 60 × oil lens. The number of LC3 puncta per cell was quantified manually. At least 20 cells in 3 independent experiments were analyzed randomly.