Ucp1 primary antibody

The brown adipose tissue homogenate of mice was mixed with RIPA lysate (Beyotime Biotechnology, shanghai, China), protease inhibitor (Roche, Basel, Switzerland), and PMSF (Beyotime Biotechnology, shanghai, China) (100:4:1), and the mixture was placed on ice for 15 min to carry out cell lysis. Then, we centrifuged the mixture and obtained the supernatant. Next, we measured protein concentration and added 5× loading buffer (Cwbiotech, Beijing, China), boiled the sample for 7 min, and loaded the proteins for electrophoresis. After electrophoresis, we transferred the proteins to a PVDF membrane. The membrane was blocked at room temperature in 5% BSA for 2 h, and the membrane was incubated with primary antibodies (UCP1 primary antibody, rabbit 1:1000, Abcam, Cambridge, UK; PGC1α primary antibody, rabbit, 1:1000, Abcam, Cambridge, UK; ACTIN primary antibody, mouse, 1:1000, Cambridge, UK; TUBULIN primary antibody, rabbit, 1:1000, Cambridge, UK) overnight at 4 °C. The following day, the membrane was washed in TBST three times, 10 min each, and the membrane was incubated with secondary antibodies for 2 h. The protein bands were visualized by chemiluminescence (Bio-Rad, Hercules, CA, USA) [34 (link)].

Tissues (adipose tissues and liver) were dissected and fixed in 10% neutral formalin for 48 h at room temperature. The fixed tissues were dehydrated in ascending series of alcohol, cleared in xylene and embedded in molten paraffin wax. Then paraffin blocks were cut into 5 mm sections and stained with hematoxylin and eosin (H&E). For IHC, sections were deparaffinated and blocked with 5% goat serum for 1 h at RT. The sections were incubated with UCP1 primary antibody (Abcam, England) overnight at 4 ℃ and anti-rabbit HRP-conjugated secondary antibody (Sangon, China) for 1 h at RT. After washing with PBS, the samples were incubated with HRP-DAB followed by nuclear staining with hematoxylin. Sections were examined by light microscopy (Abaton Scan 300/Color scanner).