The cultured peritoneal macrophages were fixed in 4% paraformaldehyde for 20 min and then washed with PBS three times. Following permeabilization with ice-cold 0.3% Triton X-100 for 10 min, the cells were blocked in 5% (m/v) bovine serum albumin (BSA, cat. no. A1993; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and then incubated with primary rabbit anti-mouse antibodies as follows: TNF-α (diluted 1:200 with PBS/5% BSA; cat. no. BS6000; Bioworld Technology, Inc., St. Louis Park, MN, USA), IL-1β (diluted 1:50 with PBS/5% BSA; cat. no. 31202; Cell Signaling Technology, Inc., Danvers, MA, USA), IL-6 (diluted 1:200 with PBS/5% BSA; cat. no. 12912; Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, cells were washed with PBS three times, followed by incubation with corresponding secondary phycoerythrin-conjugated goat anti-rabbit IgG (1:200 diluted with PBS/5% BSA; cat. no. sc-3739; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 h at room temperature. For nuclear staining, cells were covered with 10 µg/ml DAPI for 2 min at room temperature. The stained cells were visualized under a fluorescence microscope at ×400 magnification with green excitation light. Different groups of images were taken in the same software settings.
Il 1β
Manufactured by Bioworld Technology
Sourced in United States, China
IL-1β is a recombinant human interleukin-1 beta protein. It is a cytokine involved in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Bioworld Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Proliferating cell nuclear antigen (pcna), by Bioworld Technology (2 mentions)
Interleukin 6 (il 6), by Bioworld Technology (2 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
Tnf α, by Merck Group (1 mentions)
Ab36870, by Abcam (1 mentions)
Sirt1, by Abcam (1 mentions)
β actin, by Bioworld Technology (1 mentions)
β catenin, by Abcam (1 mentions)
Cyt c, by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
Cox 4, by Abcam (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
D gal, by Merck Group (1 mentions)
Pbad ser136, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
P jnk, by Abcam (1 mentions)
Psd95, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
9 protocols using il 1β
1
Immunocytochemical Analysis of Inflammatory Cytokines
2
Protein Expression Analysis in Rat Striatum
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Total protein from the perihematoma area of the rat striatum was extracted using ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with proteinase and phosphatase inhibitors. Protein concentration was determined by BCA assay. Then, a total of 50 µg protein/lane was separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for 1.5 h at room temperature in 5% non-fat-milk TBST (0.1% Tween-20) buffer and incubated overnight at 4˚C with a primary rabbit anti-rat CTRP3 (1:500; ab36870, Abcam,), SIRT1 (1:500; cat. no. BS64501, Bioworld Technology, Inc.), TNF-α (1:500; cat. no. BS6000, Bioworld Technology, Inc.), IL-1β (1:500; cat. no. BS6067, Bioworld Technology, Inc.) and β-actin (1:3,000; cat. no. AP0060, Bioworld Technology, Inc.) antibody. The membrane was incubated with secondary anti-rabbit antibody (1:4,000; horseradish peroxidase-conjugated Goat Anti-Rabbit IgG, cat. no. D110058, Sangon Biotech Co. Ltd.) for 1.5 h at room temperature. Images were captured using MicroChemi (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ version 1.8.0 (National Institutes of Health) software.
3
Colon Tissue Immunohistochemistry Analysis
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Colon tissues were collected and fixed in 4% formaldehyde overnight and stored in 70% ethanol. The fixed portion of the colon tissue was embedded in paraffin, cut into 6-μm part, and put onto the microscopic slides. Slides were either stained with hematoxylin-eosin (H&E) for histological analysis by optical microscopy or stained by immunohistochemistry for the proliferation marker PCNA, IL-1β, IL-6, TNF-α (Bioworld Technology), β-catenin (Abcam), p65 (Cell Signaling Technology), and counterstained using 3,3′-diaminobenzidine (DAB) followed by hematoxylin counterstain. Histopathology analysis was performed as previously described [47 ].
4
Detailed Protocol for Alzheimer's Research
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D-gal (Lot No. 1906754) was obtained from Sigma Aldrich Co. Ltd. (Germany, purity > 99%), and standard UB was purchased from Keygen Biotechnology Development Co., Ltd. (Nanjing, China, Lot No. 102346-214, purity > 99%). Commercial ELISA kits for monoamine oxidase (MAO, No. 20200506), acetylcholinesterase (AChE, No. 20200721), malondialdehyde (MDA, No. 20200623), superoxide dismutase (SOD, No. 20201216), total antioxidant capacity (T-AOC, No. 20200413), catalase (CAT, No. 20200326) and glutathione peroxidase (GSH-Px, No. 20200821) were purchased from Shanghai Yisheng Biotechnology Co., Ltd. (Shanghai, China). ELISA lab kits for interleukin-6 (IL-6, No. 20200625), tumor necrosis factor-α (TNF-α, No. 20200207) and interleukin-1 beta (IL-1β, No. 20200418) were purchased from Bioworld Technology Co., Ltd. (Nanjing, China). Bax, Bcl-2, cleaved caspase 3, Cu, Zn-SOD, CAT, p-p38, p38, p-JNK, JNK, Cyt C, Cox IV, GFAP, IBA1, PSD95, Syn, p-p44/42 MAPK (Thr202/Tyr204), p-p44/42 MAPK, p-AKT (Thr308), p-AKT (Ser473), AKT, Bad, P-Bad (Ser112) and P-Bad (Ser136) antibodies were all from Abcam (Shanghai, China).
5
Protein Expression Analysis in Cells
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SDS-PAGE and western blots were performed using standard protocols. Antibody binding to bands was detected using an ECL detection system (Sage Creation). The primary antibodies used were specific to MUC17 (Sigma-Aldrich, St Louis, MO, USA), IL-1β (Bioworld, St Louis Park, MN, USA), IL-8 (Bioworld), cdx1 (Abcam), pJNK (Affinity biosciences, Cincinnati, OH, USA), pERK (Santa Cruz, Dallas, TX, USA), p38 (Cell Signaling Technology, Danvers, MA, USA), pp38 (Cell Signaling Technology), PPARγ (Affinity biosciences), pp65 (ZSGB-Bio, Beijing, China), p65 (ZSGB-Bio), p21waf (Proteintech, Rosemont, IL, USA), p53 (ZSGB-Bio), Myh9 (Abcam, Cambridge, UK), and Actin (Sigma-Aldrich).
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SDS-PAGE and western blots were performed using standard protocols. Antibody binding to bands was detected using an ECL detection system (Sage Creation). The primary antibodies used were specific to MUC17 (Sigma-Aldrich, St Louis, MO, USA), IL-1β (Bioworld, St Louis Park, MN, USA), IL-8 (Bioworld), cdx1 (Abcam), pJNK (Affinity biosciences, Cincinnati, OH, USA), pERK (Santa Cruz, Dallas, TX, USA), p38 (Cell Signaling Technology, Danvers, MA, USA), pp38 (Cell Signaling Technology), PPARγ (Affinity biosciences), pp65 (ZSGB-Bio, Beijing, China), p65 (ZSGB-Bio), p21waf (Proteintech, Rosemont, IL, USA), p53 (ZSGB-Bio), Myh9 (Abcam, Cambridge, UK), and Actin (Sigma-Aldrich).
6
Protein Expression Analysis of Exosomes
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Cells, exosomes, and tissues were lysed in RIPA buffer. Protein concentration was determined using the BCA assay kit (Pierce, USA). Sources and dilution factors of primary antibodies were: rabbit polyclonal CD63 (1:1000; Bioworld, USA), CD9 (1:1000; Bioworld), CD81 (1:1000; Epitomics, USA), PCNA (1:1000; Bioworld), BCL-XL (1:100; SAB, USA), BCL-2 (1:1000; Bioworld), Bax (1:1000; Bioworld), Cytochrome C (1:500; Abcam, USA), caspase-3 (1:500; Bioworld), IL-1β (1:500; Bioworld,), LC3B (1:500; Abcam), Beclin-1 (1:600; Proteintech, USA), mTOR (1:500; SAB), p-mTOR (1:500; SAB), 4EBP1 (1:200; SAB), p70S6K (1:200; SAB), and mouse monoclonal GAPDH (1:3000; Kang Chen, China). The nucleoprotein and plasma protein was separated by the nuclear and plasma protein isolation kit (Vazyme, China), with the primary antibody NF-kB-P65 in the nucleus (1:500; SAB), primary antibody nucleoprotein Histone (1:1000; SAB). After incubation with the primary antibodies overnight at 4 °C, membranes were washed three times with Tris-buffered saline with 0.05% Tween-20 and challenged with HRP-conjugated goat anti-rabbit or goat anti-mouse antibody (1:2000; Bioworld). Western blot was performed by Luminata™ crescendo western HRP substrate (Millipore, USA) and analyzed using MD Image Quant Software.
7
Corneal Protein Expression Analysis
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Cornea tissues were collected and homogenized in RIPA (Beyotime, Shanghai, China) buffer with PMSF (Beyotime, Shanghai, China) (RIPA: PMSF = 100:1), and lysed on ice for 30 minutes. The supernatant was removed after centrifugation at 4°C (15 minutes as 12,000 rpm) and protein quantification was performed by BCA protein assay (ElabScience, Wuhan, China). Then transferred to an 0.22-µm polyvinylidene difluoride membrane (BioSharp, Tallinn, Harjumaa, Estonia) after separation by 12.5% sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The membranes were blocked with 1% skim milk powder for 1 hour, the primary antibody against β-actin (1:5000 BioWorld, Nanjing, China), tumor necrosis factor (TNF)-α (1:1000; Abcam, Branford, CT) and IL-1β (1:1000 BioWorld) were applied sequentially with overnight incubations at 4°C, and the membranes were washed three times in Tris-buffered saline with Tween. The secondary antibody was incubated for 1 hour at room temperature, washed three times with -buffered saline with Tween, and blotted with chemiluminescence (ECL; Thermo Fisher Scientific, Waltham MA) for chemiluminescence observation.
8
Testicular Protein Profiling and Oxidative Stress
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The testicular tissue samples were homogenized, and protein quantifications were performed using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein per specimen were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (0.2 μl or 0.45 μm) (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with 5% fat-free milk for 2 h at room temperature, followed by incubation with the following antibodies at 4°C overnight: nuclear factor like-2 factor (Nrf2; Abcam 1 : 1,000), HO-1 (Abcam 1 : 1,000), quinone oxidoreductase-1 (NQO-1; Abcam 1 : 1,000), NF-κB (CST 1 : 1,000), necrosis factor alpha (TNF-α) (CST 1 : 1,000), interleukin-1 beta (IL-1β) (CST 1 : 1,000), interleukin-6 (IL-6) (CST 1 : 1,000), and beta-actin (Bioworld 1 : 10,000). After washing with Tris-buffered saline and Tween 20 (TBS-T), the blots were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (BioSharp, Technology Inc., China) at room temperature for 2 h. Finally, protein bands were detected using Image Lab Software and analyzed using Image-Pro Plus software. All the experiments were repeated at least 3 times.
9
Quantitative Protein Analysis of Kidney Tissues
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About 100 mg of kidney tissue was lysated in ice-cold RIPA lysis buffer (Beyotime, China) containing 1 mM PMSF. The concentration of protein was determined by a BCA assay kit. About 50 μg protein samples were loaded in 10% SDS-PAGE gels and transferred onto the PVDF membrane (Millipore, USA). After blocking with 5% skim milk, the membrane was incubated overnight at 4 °C with the following antibodies: TGFβ1 (1:2000, Proteintech, USA), GAPDH (1:50000, Proteintech, USA), α-SMA (1:5000, Proteintech, USA), Galectin3 (1:1000, Cell Signaling, USA), p38 MAPK (1:1000, Abcam, UK), phospho-Samd2/3 (1:1000, Cell Signaling, USA), Smad2/3 (1:1000, Cell Signaling, USA), IL-6 (1:1000, Proteintech, USA), IL-1β (1:1000, Bioworld, China), and TNFα (1:1000, Proteintech, USA) antibodies. The bolts were incubated with horseradish peroxidase (HRP)-conjugated goat-anti rabbit or mouse secondary antibodies for 1 h, and the membranes reacted with chemiluminescence HRP substrate (Solarbio, China) and exposed to the ChemiScope 6000 Exp image system (CliNX, Shanghai, China) for visualization of protein bands. The protein bands were quanti ed using the NIH ImageJ Software.
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