Ab234430

Rat abdominal subcutaneous adipose tissue slices were immersed in antigen retrieval solution (P0086, Beyotime, Shanghai, China) and boiled at 95°C for 10 min to repair antigen. Ten-minute treatment with 3% H₂O₂ was then conducted to remove endogenous peroxidases in the slices. Later, the slices were blocked for 30 min using 5% bovine serum albumin (BSA; B928042; MACKLIN, China) at 37°C and incubated with primary antibody for UCP1 (ab234430; Abcam, Cambridge, UK) at 4°C overnight. Afterward, secondary antibody HRP-conjugated goat anti-rabbit IgG (31460, ThermoFisher, Waltham, MA, USA) was used to probe the slices for 1 h in the dark, followed by color development using DAB solution (D8001; Sigma-Aldrich, USA). The slices were counterstained with hematoxylin and observed via the optical microscope under 100× magnification.

The liver, epiWAT, subWAT, and interscapular BAT (iBAT) were removed from each mouse and fixed in a buffer solution of 10% formalin. Fixed tissues were routinely processed for paraffin embedding, and 4 µm sections were prepared and stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT). For immunohistochemistry (IHC) staining of subWAT and iBAT, a poly anti-Ucp1 antibody (Abcam, ab234430, Carlsbad, CA, USA) at a dilution of 1:50 was used. All stained areas were viewed using an optical microscope (Zeiss Axioscope, Jena, Germany) at a magnification of 200×.

All the tissues were fixed in 4% neutral formaldehyde for 24 h at room temperature, followed by dehydration, paraffin embedding, sectioning (4 mm), and staining with hematoxylin and eosin (H&E). To assess the fatty infiltration of skeletal muscle, tissues were snap frozen in liquid-nitrogen-cooled isopentane, sectioned at a thickness of 10 μm with a cryostat, and then stained with Oil Red O as described previously (19 (link)).
The procedure for immunohistochemistry was as previously described (20 (link)). Briefly, non-specific binding sites were blocked using 1% bovine serum albumin, followed by epitope retrieval using an autoclave (15 min in citrate buffer, pH 6.0). Then, the slides were incubated overnight at 4°C with rabbit polyclonal anti-UCP1 primary antibody (ab234430; Abcam) diluted 1:1000. After the slides were rinsed, they were incubated with HRP-conjugated goat anti-rabbit IgG (HAF008; R&D). Immunovisualization was carried out by the use of diamino-benzidine cytochemistry. The slides were finally counterstained with hematoxylin to display the nuclei. Indirect calorimetry and aerobic testing. Energy expenditure was analyzed using a Comprehensive Lab Animal Monitoring System (Columbus Instruments, United States). Pictures were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, United States). All pictures were assessed by two blinded reviewers.