Mouse tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin or oil red according to standard protocols. Immunohistochemical staining of paraffin sections was carried out with 1:50 anti-UCP-1 (Abcam), 1:50 p-CREB (Ser133) (CST), and 1:50 PKA C (CST) and detected by Inverted microscope (Olympus). The cell sizes and areas of adipose tissues were measured by Image J. Immunofluorescence staining of paraffin sections for BrdU incorporation was carried out with primary antibodies 1:50 anti-UCP-1 (Abcam), 1:100 BrdU (Santa Cruz), or 1:200 CD68 (Bio-Rad, Formerly AbD Serotec) and secondary antibodies Alexa Fluor 594 conjugated goat antibody to rabbit IgG, Alexa Fluor 488 conjugated goat antibody to mouse IgG, or Alexa Fluor 594 conjugated goat antibody to rat IgG (Invitrogen, 1:1,000). The Elecron microscopic observations were conducted through scanning electron microscope (PHILIPS CM120). All the representative images were repeated in at least 3 independent experiments.
Anti ucp1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-UCP1 is a primary antibody that specifically recognizes the Uncoupling Protein 1 (UCP1), also known as thermogenin, which is a mitochondrial inner membrane protein primarily expressed in brown adipose tissue. This antibody can be used to detect and quantify UCP1 expression in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti α tubulin, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti th, by Merck Group (3 mentions)
Anti ampk, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Alexa fluor 594 conjugated goat antibody to rabbit igg, by Thermo Fisher Scientific (3 mentions)
Anti prdm16, by R&D Systems (3 mentions)
Odyssey clx imaging system, by LI COR (3 mentions)
Anti β tubulin, by Cell Signaling Technology (3 mentions)
Anti pparγ, by Santa Cruz Biotechnology (3 mentions)
Anti ampkα, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Recombinant human bmp8, by R&D Systems (2 mentions)
Anti cd29, by Thermo Fisher Scientific (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti fasn, by BD (2 mentions)
Anti phospho hsl s660 and anti phospho perilipin, by Cell Signaling Technology (2 mentions)
Anti actin and anti tubulin, by Merck Group (2 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
59 protocols using anti ucp1
1
Histological and Immunohistochemical Analysis of Mouse Adipose Tissue
2
Multimodal Immunohistochemistry and Fluorescence Imaging
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Mouse tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin according to standard protocols. Immunohistochemical staining of paraffin sections was carried out with 1:200 anti-UCP1 (Abcam). For Immunofluorescence, slides were fixed with 4% PFA and permeabilized in ice cold methanol. Heat-mediated antigen retrieval with a 0.01 M citric acid (pH 6.0) was performed for 5 min in a microwave. After blocking with 4% BSA, the sections were blocked with unconjugated AffiniPure Fab Fragment (1:100; Jackson), then incubated with primary antibodies anti-UCP1 (rabbit; 1:200; Abcam), anti-red fluorescent protein (rabbit; 1:500; Rockland), anti-PDGFRα (goat; 1:200; R&D) or anti-DPP4 (goat; 1:200; R&D) followed by detection with secondary antibodies Alexa Fluor® 594 conjugated goat antibody to rabbit IgG (1:1,000; Invitrogen) or Alexa Fluor® 647 conjugated donkey antibody to goat IgG (1:1,000; Invitrogen). Finally, the sections were incubated with DAPI or Hoechst. The adipocytes were stained with BODIPY® 493/503 (Invitrogen). EdU incorporation assay was performed following the instruction of Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Invitrogen).
3
Adipogenic Differentiation of Stem Cells
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Recombinant human BMP7 was kindly provided by Stryker Regenerative Medicine (Hopkinton, MA), recombinant human BMP8 was purchased from R&D Systems (Minneapolis, MN). Antibody sources are as follows: anti-UCP1 was from Abcam (Cambridge, MA, ab155117) and AnaSpec (Fremont, CA, Cat # 53936); anti-α-tubulin was from Sigma-Aldrich (Dallas, TX, T6074); anti-CD29 was from eBioscience (San Diego, CA, clone TS2/16). All other chemicals were purchased from Sigma-Aldrich (Dallas, TX), unless otherwise specified.
4
Immunoblotting of Brown Adipose Tissue
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Brown adipose tissues were lysed in RIPA buffer with Complete Protease Inhibitor Cocktail (Roche Inc., Indianapolis, IN). Protein concentration was determined with BCA protein assay kit (Pierce, Rockford, IL). Twenty microgram of protein of each sample was separated by SDS-PAGE, and electro-transferred to nitrocellulose membrane for immunoblot analyses. The following antibodies were used: anti-p-PKA (Tyr197) (Cell Signaling, Danvers, MA, 4781S, 1:1000), anti-t-PKA (Cell Signaling, Danvers, MA, 4782, 1:1000), anti-p-HSL (Ser563) (Cell Signaling, Danvers, MA, 4139S, 1:1000), anti-t-HSL (Cell Signaling, Danvers, MA, 4107, 1:1000), anti-p-Creb (Ser133) (Cell Signaling, Danvers, MA, 9191S, 1:1000), anti-t-Creb (Cell Signaling, Danvers, MA, 9197, 1:1000), p-AMPKα (T172) (Cell Signaling, Danvers, MA, 2535L, 1:1000), anti t-AMPK (Cell Signaling, Danvers, MA, 2603S, 1:1000), anti-UCP1 (ABCAM, Cambridge, MA Ab10983, 1:10000), anti-β-actin (Cell Signaling, Danvers, MA, 4967S, 1:1000), HRP-conjugated anti-mouse (GE Healthcare UK Limited, 1:10,000), and anti-rabbit (GE Healthcare UK Limited, 1:10,000). The SuperSignal West Pico Chemiluminescent kit (Pierce, Rockford, IL) was used as substrates.
5
Western Blot Analysis of Adipocyte Proteins
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Tissue proteins were extracted using RIPA buffer with freshly added proteinase inhibitors. Protein concentrations were determined using BCA Protein Assay Kit (Pierce). Equal amounts of protein samples were subjected to western blot. The following antibodies were used: anti-UCP1 (Abcam, ab209483, 1:5000 dilution), anti-PGC-1a (Bioworld, BS72263, 1:500 dilution), anti-PER-ILIPIN (Cell Signaling Technology, 9349T, 1:1000 dilution), anti-PPARγ (CusAb, CSB-PA018424LA01HU, 1:500 dilution), anti-COX4 (Proteintech, 11242-1-AP, 1:1000 dilution), and anti-ACTIN (Sigma, A5441, 1:5000 dilution). Densitometry was performed using Image J. Relative band density was calculated by dividing the densitometry of target protein with loading control from the same membrane.
6
Protein Expression Analysis in iWAT
7
Western Blot Analysis of UCP1 Protein
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SAT samples were homogenized in precooled RIPA buffer containing a protease inhibitor cocktail. Following centrifugation at 12,000×g and 4 °C for 15 min, the supernatant was collected. The concentration of proteins was detected using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturer’s instructions. Equal amounts of total protein were loaded in each lane of a 10% SDS-PAGE gel, separated, and then transferred onto polyvinylidene difluoride membranes (Bio-Rad, USA). The membranes were blocked with 5% dry nonfat milk for 2 h at room temperature, probed with anti-UCP1 (Abcam, USA) or anti-GAPDH (Proteintech, China) antibodies overnight at 4 °C, and washed 5 times with phosphate-buffered saline and 0.1% Tween 20 (PBST) for 6 min each. Next, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then washed again with PBST (5 washes for 6 min each). The reaction was determined using a chemiluminescence system (ChemiDoc XRS + , Bio-Rad, USA). The band intensity was analyzed by Image Lab (Bio-Rad, USA).
8
Protein Expression Analysis in Tissues
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The tissues were homogenised in the lysis buffer (RIPA, BioTeke) containing 1 mmol/L protease inhibitor PMSF (Phenylmethylsulfonyl fluoride) (P7626, Sigma) and centrifuged at 12,000 g for 15 min. The supernatant was collected and the protein concentration was measured using BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Total protein was separated on SDS–PAGE and blotted onto PVDF (polyvinylidene fluoride) membranes, followed by incubation overnight at 4°C with the following primary antibodies: anti-AgRP (Abcam, USA, 1:1,000 dilution), anti-UCP1 (Abcam, USA, 1:5,000 dilution), anti-POMC (CST, USA, 1:1,000 dilution), and anti-β-actin (CST, USA, 1:1,000 dilution). Finally, the proteins were visualised with the Clarity Max Western ECL Substrate (Bio-Rad, #1705062S), followed by quantification of the density of bands using ImageJ software (National Institutes of Health, Bethesda, MD) and then normalisation to β-actin levels.
9
Protein Expression Analysis in iWAT
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For protein expression analyses, iWAT was homogenized in TNET buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) as described (Morley et al., 2015 (link)) with Halt protease and phosphatase inhibitors (Thermo Pierce). Samples from tissue lysates were then resolved by SDS-PAGE. Immunoblotting was performed using standard protocols. Membranes were blotted with the following antibodies: anti-FASN (BD Biosciences); anti-UCP1 (Abcam); anti-TH (Millipore); anti-phospho HSL-S660 and anti-phospho perilipin (Cell Signaling Technology), anti-Actin and anti-Tubulin (Sigma-Aldrich).
10
Western Blot Analysis of Protein Signaling
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Tissues were lysed in RIPA-buffer (Sigma–Aldrich, MO, USA) using sonicator (Diagnode, Seraing, Belgium) and centrifuged (12,000 g, 10 min, 4 °C) [21] . Proteins in supernatants were collected and blotted for anti-phospho AKTSer473 (Cat# 9271), anti-AKT (Cat# 9272), anti-phospho AMPKThr172 (Cat# 2531), anti-AMPK (Cat# 2532), anti-Actin (Cat# 12,262), anti-GAPDH (all Cell Signaling Technologies, Cell Signaling Technology, Danvers, MA, USA), anti-UCP1 (Abcam, Cambridge, UK, ab10983) and anti-GLUT1 (Dr. A. Schürmann, Potsdam) diluted in 1x TTBS+5% BSA and incubated overnight at 4 °C under gentle agitation. Subsequently, blots were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (Dianova, Hamburg, Germany) diluted 1:20,000 in 1x TTBS+5% BSA for 1h at room temperature. Specific protein bands were visualized using enhanced chemiluminescence reagents (Perkin Elmer, Waltham, MA, USA). Protein bands were quantified using Quantity One software (BioRad, München, Germany).
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