Evos x1 microscopy

The liver and fat tissues of each mouse were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into slides with a thickness of 4 μm. Liver and fat tissue sections were stained with hematoxylin and eosin (H&E) for histological analysis. For oil red O staining, liver tissues from the same liver lobe were cut into small pieces, then the frozen sections were rinsed in distilled water and stained with 0.2% oil red O (Sigma) and 60% 2-propanol (Sigma) for 10 min at 37°C. For immunohistochemistry analysis, fat slides were rinsed in 0.01 mol/L sodium citrate (pH 6.0) and heated for 20 min in a microwave to retrieve antigen. The sections were blocked in blocking buffer containing 5% goat serum, 2% BSA, 0.1% Triton X-100 and 0.1% sodium azide in PBS, then incubated overnight with anti-UCP1 (Cell Signaling) by a dilution of 1:100 at 4°C. After being washed twice in PBS, slices were incubated with secondary antibodies (Cell Signaling) for 1 h at room temperature. Slides were counterstained with H&E. All digital pictures were acquired using an EVOS X1 microscopy (Thermo Fisher Scientific).

Liver and adipose tissues were fixed in 4% paraformaldehyde, then embedded in paraffin and cut into 4-μm thick slides. The slides were stained with haematoxylin and eosin (H&E) for histology analysis. For immunohistochemistry staining, slides were antigen retrieved by submerged in 0.01 mol/L sodium citrate (pH 6.0) and heated for 20 min in a microwave. The sections were blocked in blocking buffer containing 5% goat serum, 2% BSA, 0.1% triton X-100 and 0.1% sodium azide in PBS, then incubated overnight with anti-UCP1 (Cell Signalling Danvers, USA) by a dilution of 1:100 at 4 °C. After washed twice in PBS, slices were incubated with secondary antibodies (Cell Signalling, Danvers, MA, USA) for 1 h at room temperature. Slides were counterstained with H&E and digital images were collected with an EVOS X1 microscopy (Thermo Fisher Scientific, Shanghai, China).