All specimens were set in 10% neutral formalin and embedded in paraffin, 4 μm thick sections. After dewaxing the sections, the antigen was repaired with sodium citrate buffer, washed in 1×PBS buffer, diluted 1:100 PPARγ antibody (Abcam, 41928), 1:100 VEGF-A antibody (Abcam, ab1316) and 1:100 The Vimentin antibody (Cell Signaling Technology, D21H3) was incubated overnight at 4°C, and the next day, the anti-mouse HRP secondary antibody (DAKO) was incubated at 37°C for 30 mins, and the results were observed after DAB color development. The scores were evaluated by two pathologists in the First Affiliated Hospital of Shihezi University School of Medicine. The scoring criteria were: the percentage of positive cells (0–5%, 0; 6–25%, 1; 26–50%, 2; 51–75%, 3; 76–100%, 4), positive staining intensity (negative, 0; yellow, 1; brownish yellow, 2; brown, 3), and the final total score is equal to the percentage of positive cells multiplied by the positive staining intensity.
Pparγ antibody
Manufactured by Abcam
Sourced in United Kingdom
The PPARγ antibody is a tool used in research to detect and study the peroxisome proliferator-activated receptor gamma (PPARγ) protein. PPARγ is a nuclear receptor that plays a key role in the regulation of gene expression related to energy homeostasis, cell differentiation, and inflammation.
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Lab products found in correlation
Gw9662, by Merck Group (2 mentions)
Axio observer 7 microscope, by Zeiss (2 mentions)
Vegf a antibody, by Abcam (1 mentions)
Vimentin antibody, by Cell Signaling Technology (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Lpl antibody, by Abcam (1 mentions)
C ebpα antibody, by Abcam (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Yeasen (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Beyotime (1 mentions)
Fluorescent microscope, by Leica (1 mentions)
Goat anti rabbit igg h l alexa fluor 555, by Abcam (1 mentions)
Nox4 antibody, by Abcam (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Anti rabbit igg conjugated with horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Fluromount g, by Southern Biotech (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
10 protocols using pparγ antibody
1
Immunohistochemical Analysis of PPARγ, VEGF-A, and Vimentin
2
PPAR-γ Signaling Pathway Analysis
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We purchased DHA and LA powders from Sigma-Aldrich (St Louis, MO, USA). CCK-8 kit was bought from Beyotime (Shanghai, China). The stock solution of DHA and LA was prepared in the corresponding solvent described previously. These chemical ingredients were dissolved in the complete growth medium for working solutions. PPAR-γ antibody was ordered from Abcam (Cambridge, UK). Mifobate and GW9662 were purchased from Sigma-Aldrich (St Louis, MO, USA).
3
Western Blot Analysis of Adipogenic Markers
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Protein was extracted from cultured cells by RIPA lysis buffer (Yeasen, Shanghai) with phenylmethylsulfonyl fluoride (PMSF) (1:100; Beyotime, China) and quantified with the BCA Protein Assay Kit (Beyotime). Protein lysates were separated on 15% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (0.22 μm; Millipore, Danvers, MA, USA). Then western blotting was carried out. In brief, the membranes were blocked with 5% milk at room temperature for 1 h, incubated with primary antibody overnight at 4 °C, and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000; Yeasen, Shanghai) for 1 h at room temperature. Proteins were detected by ECL reagent (Millipore). The results were obtained using a Tanon imager (Tanon, Shanghai, China). The primary antibodies used were as follows: PPARγ antibody (1:1000; Abcam), AP2 antibody (1:1000; Abcam), LPL antibody (1:1000; Abcam), CEBPα antibody (1:1000; Abcam), DSP antibody (25318-1-AP, 1:500; Proteintech, Wuhan, China), β-catenin antibody (51067-2-AP, 1:1000; Proteintech), GAPDH antibody (10494-1-AP, 1:10,000; Proteintech), and HRP-labeled ACTB antibody (1:10,000; Proteintech).
4
Immunofluorescence Staining of Cellular Markers
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The sections were stained with PPARγ antibody (1/400; Abcam, UK), p-p65 antibody (1/400; Abcam, UK), NOX4 antibody (1/400; Abcam, UK), DHE antibody (1/400; Abcam, UK), or TRIM27 antibody (1/400; Abcam, UK) at 4 °C, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) (1/1000; Abcam, UK) or Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (1/1000; Abcam, UK) for 2 h at room temperature. Subsequently, the sections were stained with a DAPI solution for 8 min at room temperature, and the images were acquired under a fluorescent microscope (Leica, Germany).
5
Western Blot Analysis of PPARs
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Hippocampal tissue (50 mg) was homogenized in 0.5 ml tissue lysate and centrifugated at 12,000 g at 4°C for 5 min. The supernatant was (20 μg protein) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% nonfat milk for 1 hour at room temperature. After washing in phosphate buffer solution (PBS) for 3 times, the membrane was incubated with a rabbit-anti-rat PPAR-α antibody (1:1000; Abcam, England) [30 ], PPAR-β antibody (1:1000; Santa Cruz, CA) [24 ], PPAR-γ antibody (1:1000; Abcam, England ) [30 ] at room temperature overnight, and then incubated with an anti-rabbit IgG conjugated with horseradish peroxidase (1:1000; Santa Cruz, CA) at 37°C for 1 hour. The color reaction was carried out using ECL reagents (Pierce, USA). A Bio-Rad imaging system was used to quantify the PPARs band.
6
Immunohistochemical Analysis of PPAR-γ
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Cryo-sections were treated with 0.3% Triton X-100 (Bio Lab Ltd.) in PBS (PBS-T) for 5 min, to permeabilize the cell membranes. The sections were then incubated in blocking solution (1% bovine serum albumin (BSA) in PBS-T) for 1 h, at room temperature. Next, sections were incubated with rabbit polyclonal anti-peroxisome proliferator-activated receptor gamma (PPAR-γ) antibody (1:200, Abcam) in PBS-T with 1% BSA, overnight, at 4 °C. After extensive washings with PBS-T 4–5 times, for 5 min each, the sections were incubated with Alexa Fluor 647 goat anti-rabbit IgG (1:400; Invitrogen) and DAPI (1:1000, Sigma) diluted in PBS-T with 1% BSA, for 2 h, at room temperature. Sections were then rinsed 4–5 times with PBS, for 5 min each, and then mounted with Fluromount-G (Southern Biotechnology) and covered with cover slips (#1.5). The slides were then imaged using a fluorescence microscope (Axio Observer 7 microscope, Zeiss).
7
Multiplex IHC Analysis of Liver Tissue
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Multiplex immunohistochemistry staining (mIHC) was performed to analyze the expression of various factors in liver tissue. Deparaffinized and hydrated tissue sections were boiled in sodium citrate buffer (pH 6.0) for antigen retrieval. mIHC was performed using a four-color immunohistochemistry staining kit (PANOVUE, 10079100020) according to the manufacturer’s protocol. Next, α-SMA antibody (1:400 dilution, #19245, CST) was incubated overnight and washed with TBS. Subsequently, 30 μL of proportionally diluted tyramide signal amplification fluorescent solution (1:100) was incubated at room temperature for 20 min. Then, the PPARγ antibody (1:300 dilution, ab59256, Abcam) was also processed following the same conditions and steps. The stained slides were scanned and quantified using the HALO Highplex FL (Indica Labs; Albuquerque, NM) analysis module of Halo software (Erber et al., 2021 (link)).
8
Evaluation of PPAR-γ Modulation in Cardiovascular Function
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Sildenafil citrate (Selleck Chemicals; cat. no. S1431), GW9662 (Abcam; cat. no. ab141125), PPARγ antibody (Abcam; cat. no. ab19481), TRPC1 antibody (Sigma-Aldrich; Merck KGaA; cat. no. T8276), TRPC6 antibody (ProteinTech Group, Inc.; cat. no. 18236-1-AP), Ki67 antibody (Cell Signaling Technology, Inc.; cat. no. 9129), β-actin antibody (Abcam; cat. no. ab8226), donkey anti-mouse IgG H&L (Alexa Fluor® 488) (Abcam; cat. no. ab150105), anti-rabbit IgG H&L (Alexa Fluor® 594) (Abcam; cat. no. ab150076), a small animal ventilator (ALC-V8D; Shanghai Alcott Biotech, Co.), and an electrophysiological recorder (BL-420F; Biosignal Acquisition and Analysis System, Chengdu Taimeng Software, Co., Ltd.) were used in the present study.
9
PPARγ Competitive Binding Assay Protocol
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GVS-12 (C20H16F3NO3, MW: 375.346, purity ≥ 98%) was designed and synthesized by our team (Fig. 1A ); LanthaScreen™ time-resolved fluorescence resonance energy transfer (TR-FRET) PPARγ competitive binding assay was purchased from Thermo Fisher Co., Ltd. (Thermo Fisher, MA, USA); fetal bovine serum (FBS) was obtained from PAA (Linz, Germany). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Rosiglitazone (a PPARγ agonist) was obtained from Sigma Co., Ltd. (St. Louis, MO, USA). STAT3, p-STAT3, suppressor of cytokine signaling 3 (SOCS3) and PPARγ antibodies were purchased from Abcam (Cambridge, UK); GAPDH monoclonal antibody was purchased from KangChen Bio-tech (Shanghai, China); horse radish peroxidase (HRP)-conjugated secondary antibodies were purchased from Abbkine (Redlands, USA). Other analytical reagent grade chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (Nanjing, China).
10
Curcumin Modulates Neuroinflammation in Alzheimer's
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Curcumin, GW9662, Aβ1–42, and Griess reagent were purchased from Sigma. Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), and Opti- Minimum Essential Medium (MEM) were producted by Gibco. PPARγ siRNA was synthesized by Invitrogen. Lipofectamine LTX and Plus Reagent was produced by Invitrogen. Choline acetyltransferase (ChAT), glial fibrillary acidic protein (GFAP), Iba-1, NF-κB p65, IκBα, and PPARγ antibodies were obtained from Abcam. IL-1β, TNF-α, and COX-2 ELISA kits were purchased from R&D Company. A choline/acetylcholine (Ach) assay kit was supplied by Abcam. A ChAT ELISA kit was obtained from MyBioSource Inc. A PPARγ transcription factor assay kit and PPARγ ligand binding domain (human recombinant) were purchased from Cayman Chemical. A co-immunoprecipitation (Co-IP) kit produced by Pierce was used, and LDH assay kit was supplied by Nanjing Jiancheng Bioengineering Institute.
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