Ab209483

BAT tissues from mice were soaked in 4% paraformaldehyde and processed routinely. After fixation for nearly 24 h, the tissues were embedded in paraffin and cut into ~5-mm-thick sections for histology and immunohistochemistry (IHC) (24 (link)). Then, these sections were mounted onto glass slides. Multiple slides were stained with Haematoxylin and eosin (H&E) for general morphologic observation. For IHC, the slides were incubated overnight at 4°C with primary antibodies against UCP1 (1:500 dilution) (ab209483; Abcam, Cambridge, United Kingdom). Immunostaining was performed with anti-rabbit horseradish peroxidase-tagged secondary antibodies, and a Diaminobenzidine Detection Kit (EliVision, Incheon, Korea) was applied for color development. Then, haematoxylin was used for counterstaining. All images were acquired using a fluorescence microscope (Imager.A2; Zeiss, Oberkochen, Germany).

UPC1 (1:5,000, Ab209483) and PGC-1α (1:1,000, Ab188102) were purchased from Abcam Co. Ltd., CA, USA. The primary antibody against b-actin (1:10000, bs-0061 R) and secondary antibodies against rabbit (1:50000, bs-0295 G-HRP) were purchased from Bioss Co. Ltd., Beijing, China. Adipose tissues were homogenized in RIPA buffer with protease and phosphatase inhibitors; the protein extracts were separated by SDSPAGE electrophoresis and transferred to a PVDF membrane. Membranes were blocked with 5% nonfat milk for 2 h at room temperature in TBST buffer (10 mM Tris, 150 mM NaCl, pH 7.6, and 0.1% Tween 20) and probed with primary antibodies overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The protein bands were developed using an ECL kit (Biosharp Co. Ltd., Anhui, China). The densitometry analysis of the bands was performed using a gel documentation system (Gel Analyzer, ShineTech, Beijing, China).

Each individual cell line was grown to a density of 6–8 × 10⁵ cells/mL and 7 × 10⁵ cells were collected. The cells used for UCP1 ChIP were first treated with 100 nM human recombinant IRISIN (# SRP8039, Sigma) for 7 days at 37 °C, 5% CO₂. After stimulation, cells were cross-linked in 1% formaldehyde for 10 min at room temperature and lysed by sonication to shear the DNA to fragments between 200 and 1000 base pairs, ensuring that the samples were kept cold (100% duty cycle for 7 × 30-s intervals). Lysates were treated overnight at 4 °C with 8 μg of UCP1 antibody (ab209483, Abcam). Protein-DNA complexes were captured on Dynabeads-Protein G (cat #, Millipore) and eluted in 1% SDS TE buffer at 65 °C. The pellets were resuspended in the appropriate buffer for PCR (95 °C 5 min, 95 °C 30 s, 56 °C 30 s, 72 °C 10 s, 72 °C 5 min, 4 °C 60 min, cycles 30) (TSE006, 2× T5 Super PCR Mix).

Brains were sliced at 30 μm thickness into four series with a sliding microtome. pSTAT3 was visualized by diaminobenzene (DAB; 34065, ThermoFisher Scientific) following treatment with Vectastain ABC kit (PK-6100, Vector Laboratories) after incubation with a biotinylated secondary antibody. BAT was formalin fixed, paraffin processed, and sectioned at 5 μm for staining with hematoxylin and eosin (H and E) or with UCP1. UCP1 staining was visualized by DAB using Bond Polymer Refine Detection kit (DS9800, Leica). Stained slides were scanned using a NanoZoomer slide scanner (Hamamatsu) and lipid droplet area was computed on H and E images using a custom app within Visiopharm version 2017.7 (Visiopharm). Primary antibodies used in this study are rabbit anti-pSTAT3 (Tyr705, 9131, Cell Signaling; 1:500) and chicken anti-GFP (ab13970, Abcam; 1:1000), rabbit anti-TRH (Dr. Eduardo A Nillni, Brown University; 1:1000), and rabbit anti-UCP1 (ab209483, Abcam, 1:4000). Secondary antibodies are donkey anti-rabbit IgG-biotin (711-065-152, Jackson ImmunoResearch Laboratories; 1:1000) and donkey anti-chicken IgY-DyLight 488 (703-486-155, Jackson ImmunoResearch Laboratories; 1:200), and donkey anti-rabbit Alexa Fluor 594 (A-21207, ThermoFisher Scientific; 1:200).

Protein was extracted from EAT, atrial tissue, adipocytes, and atrial fibroblasts and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with Tris-buffered saline with 0.1% Tween containing 5% milk and incubated overnight at 4°C with the following primary antibodies as indicated: GAPDH mouse polyclonal antibody (1 : 5000, 60004-1-Ig, Proteintech), TGF-β1 rabbit polyclonal antibody (1 : 1000, ab92486, AbCam), connective tissue growth factor (CTGF) rabbit polyclonal antibody (1 : 1000, ab6992, AbCam), collagen I rabbit polyclonal antibody (1 : 1000, ab34710, AbCam), collagen III rabbit polyclonal antibody (1 : 1000, ab7778, AbCam), UCP-1 rabbit monoclonal antibody (1 : 1000, ab209483, AbCam), FGFR1 rabbit polyclonal antibody (1 : 1000, ab10646, AbCam), FGF21 rabbit monoclonal antibody (1 : 1000, ab171941, AbCam), and PPARγ rabbit polyclonal antibody (1 : 1000, ab45036, AbCam). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence system. Densitometric analysis was performed using Scion Image software (USA).