UCP1 staining was performed on mounted paraffin-embedded sections of formalin-fixed BAT samples using UCP1 primary antibody (Abcam, Cat.No. ab10983, 1:1,000 dilution). After incubation with primary antibody at 4 °C for 24 h, the slides were washed 5 times with PBS 1X and then incubated with secondary antibody for 2 h using Vectastain Universal ABC kit (Vector Laboratories, CA, USA, Cat.No. PK-6200). After washing 3 times in PBS 1 × 10 min each, the slides were then stained and visible with 3,3'-diaminobenzidine (DAB)-peroxidase substrate solution (0.04% DAB and 0.01% H2O2).
