Ucp 1 antibody

A Western blot for uncoupling protein-1 (UCP-1) was performed as described previously [7]. Briefly, BAT adipose tissue was homogenized using the FastPrep ribolyser (MP Biomedicals) in a buffer consisting of 10 mM Na phosphate pH 7.2, 150 mM NaCl, 1% Triton, 0.1% sodium dodecyl sulfate (SDS), 0.5% Na deoxycholate, 0.2% NaN₃ and a protease inhibitor cocktail (Thermo Fischer Scientific). The BCA protein assay (Pierce) was used to determine protein concentration. An equal amount of protein was then loaded onto a 10% SDS-PAGE. Gels were transferred onto a 0.45 µm nitrocellulose membrane and blocked in 5% non-fat dry milk (Bio-Rad) in 10 mM Tris-HCl buffer with 150 mM NaCl and 0.5% Tween 20 pH 7.6 for 3 h. Hereafter, the membrane was probed with a UCP-1 antibody (Sigma-Aldrich). For the loading control, β-actin was used. A horseradish peroxidase-conjugated secondary antibody in TBST containing 5% non-fat dry milk was then added. Signals were detected using enhanced chemiluminescence (Thermo Fischer Scientific). Analysis was performed with Image J (NIH).

For H&E staining, tissues were fixed with 10% formalin and paraffin-embedded, and then standard procedures were performed. To label blood vessels in vivo, Fluorescein labeled Griffonia Simplicifolia Lectin I (4 µg per mouse, Vector Laboratories, Cat# FL-1101) was gently injected into 2-month-old male mice via tail vein. After 20 min, the mice were sacrificed and iWAT tissues were isolated and carefully minced with scissors. The iWAT tissues were washed twice with pre-warmed PBS and cover-slipped by Fluoromount-G (SouthernBiotech, Cat#0100-01). For immunofluorescence staining, formalin-fixed and paraffin-embedded tissue sections were deparaffinized and rehydrated through graded ethanol solutions. After pre-incubation with a blocking buffer (PBS containing 5% normal goat serum and 0.3% Triton X-100) for 60 min, slides were incubated with Ucp1 antibody (Sigma, Cat#U6382) (1:500 dilution), Tom20 antibody (Santa Cruz, Cat#sc-17764) (1:100 dilution), or CD31 antibody (1:250) (Millipore Cat#MAB2148-C) in blocking buffer at 4 °C overnight. Subsequently, the slides were washed, and incubated with Alexa Flour 488-conjugated or 594-conjugated secondary antibody and DAPI for 60 min. Images were acquired and processed with the same setting for transgenic mice or knockout mice and control littermates.