Lsm880 laser scanning confocal microscope

After euthanasia, sWAT and visceral WAT (vWAT) were rapidly collected and fixed in 4% paraformaldehyde solution for 48 h. The tissues were then paraffin-embedded and the resulting blocks were cut into 5–10 µm sections and stained with hematoxylin and eosin (H&E) to assess adipose histology. Photomicrographs were obtained using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan).
For immunofluorescence analysis, fixed cells and WAT sections were stained with rabbit anti-UCP1 (dilution, 1:200) or anti-CPT1 (dilution, 1:1000) antibodies overnight at 4 °C in a moist chamber. Fluorescein isothiocyanate (FITC)-conjugated (dilution, 1:1000) and Alexa Fluor™ 594-conjugated (dilution, 1:1000) secondary antibodies were used. Mitochondria were identified by staining with 1 mM MitoTracker Red (Cell Signaling Technology, Danvers, MA, USA), according to the manufacturer’s protocol, and nuclei were stained using DAPI (Thermo Fisher Scientific, Waltham, MA, USA) fluorescence. After mounting using ProLong Gold Antifade reagent (Thermo Fisher Scientific), fluorescence images were captured using a Zeiss confocal laser scanning microscope (LSM880; Carl Zeiss, Oberkochen, Germany) and Zen 2012 software (Carl Zeiss).

Samples of VAT, QUA, and GAS were deparaffinized and incubated with anti-PKA, anti-UCP1, anti-MyoD, or anti-UCP3 antibodies. Secondary anti-mouse fluorescein isothiocyanate (FITC)-conjugated and anti-rabbit Alexa Fluor™ 594-conjugated antibodies were then applied. DAPI (Thermo Fisher Scientific, Waltham, MA, USA) was used to stain the cell nuclei. Fluorescent images were captured using a Zeiss confocal laser scanning microscope (LSM880; Carl Zeiss, Oberkochen, Germany) with Zen 2012 software (Version 1.1.1.0, Carl Zeiss).

WAT sections were deparaffinized and then incubated with anti-PKA or anti-UCP1 antibodies. Secondary anti-mouse fluorescein isothiocyanate (FITC)-conjugated and anti-rabbit Alexa Fluor 594-conjugated antibodies were then applied. DAPI (Thermo Fisher Scientific, Waltham, MA, USA) was used to stain the cell nuclei and the sections were then mounted using ProLong Gold Antifade reagent (Thermo Fisher Scientific). Fluorescent images were captured using a Zeiss confocal laser scanning microscope (LSM880; Carl Zeiss, Oberkochen, Germany) and Zen 2012 software (Version 1.1.1.0, Carl Zeiss).

Differentiated cells were seeded onto poly-L-lysine pre-treated cover slips (12 × 12 mm) and fixed using 4% paraformaldehyde for 20 min. For mitochondrial staining, 1 mM MitoTracker Red (Cell Signaling) was added before the fixation, according to the manufacturer’s protocol. The cells were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated with rabbit anti-UCP1 (1:500 dilution) antibody overnight at 4 °C. Alexa Fluor™ 594-conjugated and fluorescein isothiocyanate (FITC)-conjugated (1:1000 dilution) secondary antibodies were then applied, and the cell nuclei were stained using DAPI (Sigma-Aldrich). Finally, the cells were mounted using ProLong Gold Anti-fade reagent (Thermo Fisher Scientific, Waltham, MA, USA), and images were obtained using a Zeiss confocal laser scanning microscope (LSM880; Carl Zeiss, Oberkochen, Germany) and 2012 software (Carl Zeiss).

WAT and gastrocnemius muscle in hindlimb samples were rapidly collected and fixed in 4% paraformaldehyde solution for 48 h. The tissues were then paraffin-embedded and the resulting blocks were cut into 10 µm sections and stained with hematoxylin and eosin (H&E) to assess the histology. Photomicrographs were obtained using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan).
For immunofluorescence analysis, the tissues were fixed and stained with rabbit-GLUT4 (dilution, 1:500), -UCP3 (dilution, 1:200), or -MYH3 (dilution, 1:1000) antibodies overnight at 4 °C in a moist chamber. Alexa Fluor™ 594-conjugated and fluorescein isothiocyanate (FITC)-conjugated (dilution, 1:500) secondary antibodies were used, and DAPI (Sigma Aldrich, St. Louis, USA) was used to stain the cell nuclei. After mounting using ProLong Gold Antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA), fluorescent images were captured using a Zeiss confocal laser scanning microscope (LSM880; Carl Zeiss, Oberkochen, Germany) and Zen 2012 software (Carl Zeiss).