For SF-1 staining, brain sections were mounted on Superfrost Plus microscope slides (Fisher Scientific), then dried overnight. The following day, samples were pretreated with 0.3% hydrogen peroxide in PBS (pH 7.4) for 30 minutes at RT; then antigen retrieval was done for 5 minutes. Samples were blocked in 3% normal donkey serum, then incubated with rabbit anti–SF-1 antibody (22, 52) overnight at 4°C. After washing in PBS, sections were incubated in biotinylated donkey anti-rabbit IgG (065-152, 1:1000, Jackson ImmnuoResearch) for 2 hours at RT, followed by incubation for 1 hour in a solution of avidin-biotin complex (Vectastain Universal ABC Kit, PK-6200, Vector Laboratories). Sections were next washed in PBS and stained with DAB-peroxidase substrate solution (0.04% DAB and 0.01% H2O2) in PBS. The stained slides were visualized by a Nikon Digital Camera DXM1200 microscope system.

For UCP1 staining, iBAT was sectioned and paraffin embedded. The sections were incubated with primary UCP1 antibody (catalog ab10983, 1:1000, Abcam) at 4°C for 24 hours. Slides were washed 5 times with PBS and then incubated with secondary antibody for and additional 2 hours using the Vectastain Universal ABC Kit. After washing 3 times in PBS for 10 minutes each, slides were then stained with DAB-peroxidase substrate solution. Stained slides were visualized by a Nikon Digital Camera DXM1200 microscope system.

Peripheral tissues including WAT, BAT, liver, pituitary, adrenal gland, testis, ovary, and femurs were dissected and post-fixed overnight at 4°C. Tissue samples were then paraffin embedded and cut into 4 μm slices. Slices were stained with H&E following the standard H&E procedure.

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