UCP1 immunocytochemistry was performed as previously described,10 using a 1:500 solution of anti‐UCP1 antibody (Abcam, Cambridge, UK) and a 1:500 dilution of Alexa‐633‐conjugated secondary antibody. Samples were mounted with FluoroGel mounting medium and examined with a Zeiss Elyra PS.1 microscope. Relative fluorescence intensities were measured with ImageJ software (https://imagej.nih.gov/ij/ ).
Anti ucp1 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The Anti-UCP1 antibody is a laboratory reagent used to detect and quantify the expression of the Uncoupling Protein 1 (UCP1) in biological samples. UCP1 is a mitochondrial protein primarily found in brown adipose tissue and is involved in thermogenesis. This antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of UCP1 in research settings.
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Anti prdm16 antibody, by R&D Systems (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Pparγ, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit igg h l hrp conjugate, by Bio-Rad (2 mentions)
Ripa buffer, by Boston BioProducts (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Mouse diabetes multiplex assay kit, by Bio-Rad (2 mentions)
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Oligo dt 12 18 primer, by Thermo Fisher Scientific (2 mentions)
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Hf diet, by Research Diets (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
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Dulbecco s modified eagle s medium dmem, by Merck Group (2 mentions)
36 protocols using anti ucp1 antibody
1
UCP1 Immunocytochemistry Visualization
2
Protein Expression Analysis in Brown Adipocytes
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Whole-cell lysates from frozen tissues and brown adipocytes were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer (150 mmol/L Tris-HCl, 50 mmol/L NaCl, 1% NP-40, 0.1% Tween-20). Protease and phosphatase inhibitors were added to all buffers before experiments. Western blot was performed as previously described [21 (link)]. Protein concentrations were assayed using a Quick StartTM Bradford Assay (Bio-Rad, Hercules, CA, USA). The primary antibodies anti-phosphorylated AMPK (p-AMPK) antibody (Cell Signaling, Danvers, MA, USA), anti-PPARγ antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PGC-1α, and anti-UCP1 antibody (Abcam, Cambridge, UK) were incubated overnight at 4°C, and specific proteins were visualized by the WESTSAVEupTM Detection system (AbFrontier, Seoul, Korea). Band intensities were measured using the GeneTool (SynGene, Cambridge, UK) and normalized to β-actin.
3
Evaluating UCP1 and Mitochondrial Biogenesis
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Immunofluorescence was used to evaluate the effects of BG and Rb1 on fundamental protein, UCP1, and mitochondrial biogenesis using both 3T3-L1 cells and PWATs. For immunocytochemical analyses, differentiated adipocytes were fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA), followed by blocking with 1% BSA in PBST for 30 min. After that, the cells were incubated with anti-UCP1 antibody (1:250, Abcam) overnight at 4 °C. The cells were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody (1:500, Promega, Madison, WI, USA) in 1% BSA for 1 h. Mitochondria were stained using MitoTracker Red CMXRos (500 nM; Cell Signaling Technology) for 30 min according to the manufacturer’s protocol. Then, the cells were fixed and rinsed three times with PBS. The nuclei of the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, MA, USA). Fluorescence images were captured by using an inverted phase-contrast microscope with fluorescence (KI-2000F, Korea Lab Tech, Gyeonggi, Korea) and analyzed with image-processing software (OptiView 3.7, Korea Lab Tech, Gyeonggi, Korea).
4
Quantification of Ucp1 Protein in BAT
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The Ucp1 protein content in BAT was determined by Western blot. Tissues were homogenized in RIPA (radio immunoprecipitation assay lysis buffer), and the protein was extracted and stored at − 20 °C. The protein content was quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA) following manufacturer’s instructions.
First, 15 µg of protein in Laemmli loading buffer was denatured, loaded onto 10% acrylamide gels made with TGX™ Fast Cast™ Acrylamide Solutions (Bio-Rad, Barcelona, Spain) and run at 90 V for 75 min. Gels were then transferred onto a PVDF membrane using the Trans-Blot Transfer System (Bio-Rad, Barcelona, Spain) with Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Barcelona, Spain) following the manufacturer’s instructions. The membrane was blocked and then incubated with anti-Ucp1 antibody (Abcam, Cambridge, United Kingdom) at 4 °C overnight. Afterwards, the membrane was incubated for 2 h with the secondary antibody (GE Health Care Life Sciences, Barcelona), and the protein was detected with the chemiluminescent reagent ECL Select Western Blotting Detection Reagent (GE Healthcare, Barcelona, Spain). Protein levels were quantified with the open source software ImageJ [22 (link)] and normalized to β-actin protein levels.
First, 15 µg of protein in Laemmli loading buffer was denatured, loaded onto 10% acrylamide gels made with TGX™ Fast Cast™ Acrylamide Solutions (Bio-Rad, Barcelona, Spain) and run at 90 V for 75 min. Gels were then transferred onto a PVDF membrane using the Trans-Blot Transfer System (Bio-Rad, Barcelona, Spain) with Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Barcelona, Spain) following the manufacturer’s instructions. The membrane was blocked and then incubated with anti-Ucp1 antibody (Abcam, Cambridge, United Kingdom) at 4 °C overnight. Afterwards, the membrane was incubated for 2 h with the secondary antibody (GE Health Care Life Sciences, Barcelona), and the protein was detected with the chemiluminescent reagent ECL Select Western Blotting Detection Reagent (GE Healthcare, Barcelona, Spain). Protein levels were quantified with the open source software ImageJ [22 (link)] and normalized to β-actin protein levels.
5
Quantifying Lipid Droplets and Immune Cells
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The areas and size of lipid droplets that had accumulated in the liver and adipose tissues were measured using light microscope and image analysis software for quantification (Image J software v. 1.50i; National Institutes of Health, Bethesda, MA, USA; https://imagej.nih.gov/ij/download.html ) [34 (link)]. Immunofluorescence staining of CD68 in the abdominal visceral adipose tissue was performed using an anti-CD68 antibody (1:200) (Abcam). Texas Red X-conjugated goat anti-mouse IgG and Alexa 488-conjugated goat anti-rabbit IgG (1:500) (Invitrogen, Grand Island, NY, USA) were used as secondary antibodies. Sections were mounted and images acquired using fluorescence microscopy (IX81, Olympus, Tokyo, Japan). For immunologic staining of UCP1, abdominal subcutaneous fat was fixed with formalin and paraffin wax-embedded for immunohistochemistry. Sections were immunostained with an anti-UCP1 antibody (1:40,000) (Abcam) and 3,3′-diaminobenzidine (DAB).
6
Immunohistochemical Analysis of UCP1 in Adipose Tissue
7
Histological Analysis of Adipose Tissue
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Adipose tissues were fixed in 4% formaldehyde overnight. After paraffin embedding and sectioning at 5 µm, the sections were stained with hematoxylin and eosin (Beyotime) according to standard protocols. For immunohistochemistry, the sections were blocked with 5% BSA in PBS for 1 h, and incubated with anti‐PRDM16 antibody (R&D systems) at 4 °C overnight. The results were imaged with a light microscopy Motic BA600 (Motic Germany GmbH) and a slice scanner Pannoramic MIDI II (3DHISTECH). PRDM16‐Luc SVCs were differentiated into adipocytes and treated with DHT (1 µm , Meilunbio) or DMSO for 24 h. The adipocytes were washed with PBS, fixed in 4% formaldehyde for 10 min, permeabilized in PBST (PBS +0.1% TritonX‐100) for 10 min, blocked in 5% BSA for 30 min on ice, and incubated with Anti‐UCP1 antibody (Abcam) at 4 °C overnight. The 2nd day, after washing for three times, the adipocytes were incubated with fluorescently labeled goat anti‐rabbit IgG (Thermo Fisher Scientific) for 1 h, followed by incubation with BODIPY 493/503 (Thermo Fisher Scientific) plus Hoechst 33342 (Merck) for 20 min. Laser scanning microscopy 800 (ZEISS) with oil‐immersion objective lens was used for confocal imaging.
8
Immunoblotting for Protein Detection in Fat Tissues
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Immunoblotting for protein detection was conducted as we described [27 (link),32 (link)]. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Tissue lysates were resolved by SDS-PAGE gels. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). Primary antibodies used were as follows: Anti-UCP1 antibody (1:500, ab23841, Abcam, Cambridge, MA, USA); Anti-α-Tubulin antibody (1:1000, ABCENT4777, Advanced BioChemicals, Lawrenceville, GA, USA); Mitochondrial total OXPHOS protein antibody set (Abcam,ab110413); and Anti-pHSL (4126s, Cell Signaling Technology, Danvers, MA, USA); DNMT3b (sc-393845, Santa Cruz, Dallas, TX, USA, sc-393845); HSL (Cell Signaling, 4107s).
9
UCP1 and PKA Substrate Antibody Protocol
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Chemicals, unless stated otherwise, were purchased from Sigma–Aldrich. Anti-UCP1 antibody was purchased from Abcam, and anti-PKA substrate antibody was from Cell Signaling Technology. The oligonucleotides sequences used for various experiments are listed in Table S5 .
10
Histological Analysis of Adipose Tissue
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Epididymal, inguinal subcutaneous and retroperitoneal fat tissues were fixed in 10% formalin, processed and paraffin-embedded. Multiple sections (5 μm) were prepared and stained with hematoxylin and eosin (H&E) for general morphological observation. For immunocytochemistry staining, the sections of VAT were incubated with anti-UCP1 antibody (Abcam, MA, USA) for 30 min at room temperature. The signals were detected using a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, CA, USA) in combination with the ABC kit (Vector Laboratories) and DAB substrate (Vector Laboratories). Samples were visualized using a Nikon Eclipse 80i upright microscope (Nikon, Tokyo, Japan).
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