Rabbit anti ucp1

Samples of BAT and subcutaneous WAT were dissected, immediately fixed with ice-cold 4% (w/v) paraformaldehyde in PBS, and embedded in paraffin, and sections of 4 μm were obtained. The sections were deparaffinized at 60 °C for 20 min, immersed in xylene, rehydrated through graded ethanol solutions, and finally in distilled water. The sections were washed with 1X PBS and blocked with 10% rabbit serum (Santa Cruz Biotechnology) for 30 min at room temperature. The sections were subsequently incubated with rabbit anti-UCP1 1:100 (Abcam) at room temperature for 1 h. After washing with 1X PBS, the sections were incubated with goat anti-rabbit FITC-conjugated secondary antibody (1:500; Santa Cruz Biotechnology) at room temperature for 1 h. The sections were washed again with 1X PBS, mounted with UltraCruz™ mounting medium (Santa Cruz Biotechnology), and viewed on a Leica DM750 microscope (Leica, Wetzlar, Germany).

The primary antibodies used were: a rabbit anti-phospho-STAT3 (Tyr705) (D3A7) (1:1000; Cell Signaling, Danvers, MA, USA; 9145), a rabbit anti-STAT3 (D1A5) (1:1000; Cell Signaling, Danvers, MA, USA; 8768), a mouse anti-Agrp (1:2000; Alpha Diagnostics, San Antonio, TX, USA; AGRP-11S), a rabbit anti-Pomc (27–52, porcine) (Phoenix Pharmaceuticals, Burlingame, CA, USA; H-029-30), a mouse anti-β-actin (8H10D10) (1:5000; Cell Signaling, Danvers, MA, USA; 3700S), and a rabbit anti-UCP1 (1:500; Abcam, Cambridge, MA, USA; ab10983).

White adipose tissue (WAT) was homogenized in cell lysis buffer and extracted as previously described (Park et al. 2015). Renal tissues were lysed and subsequently sonicated in PBS that contained 1% Triton x‐100, 250 μmol/L phenylmethanesulfonyl fluoride (PMSF), 2 mmol/L EDTA, and 5 mmol/L dithiothrietol (DTT) (pH 7.5). Protein concentrations were determined by the use of Coomassie reagent. Forty microgram of protein for each sample was denatured in boiling water for 10 min, then separated by SDS‐PAGE, and transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% nonfat dry milk in Tris‐buffered saline (TBS), followed by incubation overnight with primary antibody. After washing with TBS, blots were incubated with goat anti‐rabbit/mouse horseradish peroxidase (HRP)‐conjugated secondary antibody and visualized using Enhanced Chemiluminescence (ECL). Densitometry was quantified using Image‐Pro Plus (Media Cybernetics). Primary antibodies included: goat anti‐AQP2 antibody (Cat#sc‐9882,Santa Cruz Inc, Dallas, TX), and rabbit anti‐UCP1 (Cat# ab10983, Abcam Inc, Cambridge, MA). Renal AQP2 was normalized using β‐actin as a protein loading control. For immunoblotting of UCP1 in apididymal fat, Ponceau red staining was performed to validate equal loading.

WAT, BAT, and liver samples were fixed in 10% formalin buffer for 24 h, and then dehydrated and embedded in paraffin by a standard procedure. Sections of 3 μm were prepared with a microtome and stained using a standard Hematoxylin/Eosin Alcoholic (BioOptica) procedure according to the manufacturer's instructions^{73 (link)}. Alternative sections of paraffin were used for immunohistochemistry detection of UCP-1. Immunohistochemistry was performed as described^{24 (link),79 (link)} using a rabbit anti-UCP-1 (1:2000; Abcam; Cambridge, UK). UCP-1-positive cells were counted by using Frida Software (the Johns Hopkins University; Baltimore, MD, USA). Up to 10 animals per experimental group were used and 3 pictures per each image sections were analyzed.

PAT from control and ischemic mice were harvested after PFA 4% perfusion. Paraffin cross-sections (5 µm) were cut. Dewaxed sections were incubated with rabbit anti‐UCP1 (Abcam, Cambridge, UK), according to the manufacturer’s protocol. Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol and incubated with normal rabbit serum (1:75) for 20 min to reduce nonspecific background. The tissue sections were incubated with primary antibodies against UCP1 overnight at 4°C. After incubation with secondary antibody (IgG biotin conjugated (1:200; Vector Laboratories, Newark, CA, USA), enzymatic reaction was performed to reveal peroxidase with Sigma Fast 3,3′‐diaminobenzidine (Sigma‐Aldrich, St. Louis, MO, USA) as substrate. Finally, sections were counterstained with hematoxylin and mounted.
For fibrosis quantification, paraffin cross-sections (8 µm) of infarcted hearts were cut, fixed in Bouin solution (Sigma-Aldrich) and stained with Masson’s trichrome (Sigma-Aldrich), according to the manufacturer’s protocol. Fibrosis area was quantified as the relative area of blue staining (collagen) compared to the left ventricle surface, as an average of three sections per heart at different levels, using ImageJ software.

Immunohistochemistry was performed as described [40 (link), 41 (link)]. Briefly, tissue slides of BAT and inguinal fat were dewaxed in xylene, rehydrated in ethanol (100%, then 95%, ethanol washes) and rinsed in PBS. A heat-induced antigen retrieval step with Citric Acid Based Antigen Unmasking Solution (Vector laboratories, Burlingame, CA) was used to unmask antigens. To block endogenous peroxidases, slides were incubated in 3% H₂O₂ for 30 min at room temperature and then rinsed in PBS. Before primary antibody was applied, slides were soaked in blocking solution (containing 5% sheep serum, 0.2% BSA, and 0.1% Triton X-100 in PBS) for 1 h at room temperature. The following antibodies were used: rabbit-anti UCP-1 (1:50; Abcam) and mouse- anti mitochondria (1:25; Abcam). All antibody staining was performed at 4 °C overnight, followed by incubation with 1:1000 diluted anti-biotin secondary antibody (Vector Laboratories) for 45 min at room temperature. Slides were developed using a DAB kit (Vector Laboratories) and imaged using a DS-Fi1 camera connected to a Nikon E80i stereomicroscope. Images were processed using Nikon imaging software, NIS Elements RA3.2.

Tissue fragments were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS using ball bearings and a TissueLyser (Qiagen). Lysis buffer was supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher). Protein lysates were quantitated using Pierce BCA protein assay (Thermo Fisher). Western blotting was performed using Miniprotean TGx 4–20% SDS-PAGE gels, Tetra Cell rig, and Transblot Turbo transfer system (Biorad). Membranes were blocked with 5% milk in 1× TBS-T, before overnight incubation with primary antibodies. HRP-conjugated secondary antibodies against rabbit- and mouse-derived primary antibodies were used at 5000-fold dilution and were obtained from Cell Signaling Technologies (7074S, 7076S). Chemiluminescence was visualized and recorded digitally using the Chemidoc XRS + Imaging System (Biorad). Primary antibodies used in this study include: rabbit anti-Cdkal1 (abcam, ab68045), mouse anti-ANT1 (abcam, 110322), rabbit anti-MMS19 (Proteintech, 16015-1-AP), mouse anti-total OXPHOS rodent cocktail (abcam, ab110413), mouse anti-FAM96B (Santa Cruz, sc-376801), mouse anti-FLAG tag (Sigma, F1804), mouse anti-FLAG M2 HRP-conjugated (Sigma, A8592), rabbit anti-UCP1 (abcam, 23841), and rabbit anti-β-Tubulin (Cell Signaling, 2146).