Lysates (60 μg) were extracted from the tumor tissues of mice, and were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) then transferred onto polyvinylidene difluroide membrane (PVDF, Millipore, USA). The membranes were blocked with 5% non-fat milk, followed by incubated with appropriate primary antibodies (anti-XIAP, anti-VEGF, anti-Bcl-2, anti-cyclin D1, anti-MMP-9, anti-COX-2, anti-TNF-α, anti-RANKL, anti-OPG, anti-t-ERK, anti-p-ERK, anti-cytochrome C, anti-caspase 3 and anti-caspase 8 all purchased from Millipore) at 4 °C overnight with gentle shaking. A secondary peroxidase-conjugated anti-rabbit or anti-mouse antibody was diluted at 1:1000, followed by incubation for 1 hour at room temperature. The membranes were subjected to an enhanced chemiluminescence system, and immunoreactive bands were captured on the photographic film. The Image J software (National Institutes of Health, Bethesda, USA) was used for quantification. The experiments were repeated three times.
Anti tnf α
Manufactured by Merck Group
Sourced in United States
Anti-TNF-α is a laboratory product for use in research applications. It functions as an antibody that binds to and neutralizes the tumor necrosis factor alpha (TNF-α) protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2, by Merck Group (3 mentions)
Anti caspase 3, by Merck Group (2 mentions)
Anti mmp 9, by Merck Group (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti bax, by Merck Group (2 mentions)
Anti il 6, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti vegf, by Merck Group (1 mentions)
Anti cyclin d1, by Merck Group (1 mentions)
Anti cytochrome c, by Merck Group (1 mentions)
Anti caspase 8, by Merck Group (1 mentions)
Anti p erk, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti gap43, by Merck Group (1 mentions)
Fitc conjugated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
Eclipse te300, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Anti mif, by Santa Cruz Biotechnology (1 mentions)
12 protocols using anti tnf α
1
Protein Expression Analysis in Tumor Tissues
2
Immunofluorescence Staining of Frozen DRG Sections
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Frozen DRG sections (8 µm) were fixed with 4% paraformaldehyde for 30 min. Following blocking with 5% bovine serum albumin, the DRG sections were incubated with anti-TNF-α (Millipore; 1∶100) and anti-GAP43 (Millipore; 1∶100) antibodies overnight at 4°C. Following 3 washes in 1× PBS, the sections were incubated with an anti-rabbit Alexa Fluor 594-conjugated secondary antibody (Molecular Probe; for TNF-α, 1∶200) or an anti-mouse FITC-conjugated secondary antibody (Dako, for GAP-43, 1∶200) for 1 hour at room temperature. The sections were then washed twice in 1× PBS and incubated for 5 min with 4′,6-diamidino-2-phenylindole (DAPI; 10 µg/ml; Sigma) for nuclear counterstaining, followed by 3 additional washes with 1× PBS. Finally, the sections were dried, mounted and observed through fluorescence microscopy (Nikon, Eclipse, TE300). After staining, images of samples from three independent experiments were captured on a fluorescence microscope. Ratios of fluorescent signal were measured from three random of microscopic areas (18548.61 µm2 per field) [23] (link). Images were captured using a digital camera (EvolutionTM VF) and analyzed with Image-Pro Plus® software (Media Cybernetics, Silver Spring, MD).
3
Immunoblot Analysis of Ocular Proteins
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Three to five individual eyecups, without the lens and cornea, from each group were homogenized, centrifuged, and 50 µg of the soluble protein were loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. The proteins were detected with the following primary antibodies: mouse anti-IL-1β (1:1,000, Millipore), rabbit anti-IL-6 (Proteintech, 1:1000), anti-TNF-α (1:1,000, Millipore) and anti-MIF (1:1000, Santa Cruz), mouse anti-rhodopsin (1D4, 1:4,000), rabbit anti-M-opsin (1:1,000, Millipore), goat anti-S-opsin (1:1,000, Santa Cruz), and rabbit anti-caspase 3 (1:1,000, Cell Signaling Technology). After stripping, the same membranes were probed with rabbit anti-actin-HRP (Horseradish peroxidase conjugate; 1:1,000, Cell Signaling Technology) or rabbit anti-GAPDH (1:2,500, Abcam). Development of bands, image capture, and the densitometric analysis of the bands were the same as we previously reported [5 (link)].
4
Western Blotting of Macrophage Proteins
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Whole lysates of THP-1-Mφ and iPS-Mφ were extracted with a protein extraction kit according to the manufacturer’s instructions. Western blotting was performed following standard methods. Detailed antibody information is provided below: anti-Actin (Sigma-Aldrich, dilution ratio 1:1000), anti-TNF-α (Sigma-Aldrich, dilution ratio 1:1000), anti-apoptosis-related protein cysteine-3 (Caspase-3) (Sigma-Aldrich, dilution ratio 1:1000), and anti-B-cell lymphoma-2 (Bcl-2) (Sigma-Aldrich, dilution ratio 1:1000).
5
Sciatic Nerve Protein Analysis
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Proteins expressed in the sciatic nerve were determined by western blot analysis. Sciatic nerves were rapidly dissected from rats and used for protein analysis. All the procedures including protein extraction, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and western blotting were essentially the same as described in our previous report [8 (link)]. Briefly, cells in the sciatic nerves were lysed using triton lysis buffer, sonicated, and centrifuged to collect the supernatant containing proteins. Protein (15 μg) was used for SDS-PAGE (10%) and western blotting analyses. Anti-phospho-Erk1/2 (1 : 1250, rabbit polyclonal, Cell Signaling, Seattle, USA), anti-p38 (1 : 1250, rabbit polyclonal, Santa Cruz Biotech.), anti-TNF-α (1 : 1250, rabbit polyclonal, Sigma), and anti-actin (1 : 10,000, mouse monoclonal, MP Biomedicals, Santa Ana, USA) antibodies were used as primary antibodies, and horseradish peroxidase- (HRP-) conjugated antibody (1 : 1250; goat anti-rabbit or goat anti-mouse, Santa Cruz Biotech.) as a secondary antibody. Protein band intensity in the scanned images of X-ray film was determined by using the i-Solution software (Image & Microscope Technology, Daejeon, Korea).
6
Western Blot Analysis of Protein Signaling
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After two washes with phosphate buffered saline (PBS), cells were scrapped on ice into lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA) supplemented with 1 mM PMSF and the soluble fraction was isolated after centrifugation (14000 rpm, 10 min, 4°C). Protein was quantified using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s protocol, and lysates were resolved on 8% poly-acrylamide gels and transferred onto Immobilon-P PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% milk in Tris buffered saline with 0.1% Tween-20 (TBST) for 1 hr, followed by an overnight incubation at 4°C in primary antibody (1:1000). Membranes were washed with TBST before and after exposure to goat-anti-rabbit HRP secondary antibody (1 hr; Cell Signaling) and protein were visualized using Kodak 1D Image Analysis Software 3.6 and a Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Primary antibodies used for western blotting include phospho-p44/42 MAPK (ERK1/2), p44/42 MAPK (ERK1/2), phospho-AKT, AKT, phospho-JNK, phospho-IKK, phospho-NF-κB, phospho-elF-2α, and phospho-TAK1 were obtained from Cell Signaling, anti-GPR120 was obtained from Abcam and anti-β-actin and anti-TNFα were obtained from Sigma.
7
Protein Expression Analysis in THP-1 Cells and HVSMC
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Proteins were extracted from THP-1 transfected with miR-155 mimic or miR-155 inhibitor; HVSMC treated with supernatant of the transfection cells as above and tumor tissue from mouse. Western blotting was performed to assess the rabbit anti-MMP2 antibody (1:1000; Thermo Scientific, U.S.A.), goat anti-MMP9 antibody (1:500; Santa Cruz Biotechnology, U.S.A.), rabbit anti-monocyte chemoattractant protein (MCP-1) antibody (1:500; Bio-Rad Laboratories, U.S.A.), anti-iNOS antibody (1:100, Santa Cruz), anti-TNFα (1:1000, Sigma-Aldrich), anti-α-SMA antibody (1:1000; Millipore U.S.A.), and anti-Osteopontin antibody (1:500; Millipore, U.S.A.). Briefly, proteins (30 µg) were separated using 12% SDS gel electrophoresis and electrotransferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, U.S.A.). Then membranes were blocked with 5% nonfat dry milk in TBS-Tween for 1 h at room temperature, and incubated with primary antibodies at 4°C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibody was used at a 1:5000 dilution (DAKO) for 2 h at room temperature. Bands were visualized using enhanced chemiluminescence (ECL Advance™; GE Healthcare). Protein expression was quantified with densitometric analysis using the ChemiDoc™ imaging system (Bio-Rad Laboratories) and QuantityOne™1-D Analysis Software (Bio-Rad Laboratories).
8
Quantifying Immune Markers in the Stratum Corneum
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Biologically active substances that could not be quantified by ELISA due to their low amounts in the stratum corneum were measured by immunocytochemistry. According to a previously described method,20 sample tape strips were pasted onto a glass slide to transfer the corneal cells by immersing them in xylene overnight. The cells were subsequently fixed with cold acetone, washed with PBS containing 0.3% Triton X‐100 (PBST), and blocked with 0.3% Triton X‐100 and 1% BSA containing 0.1% NaN3 (1% BSA in PBST; Vector Laboratories). The primary antibody was added overnight at 4°C, and samples were washed with PBST and then reacted at room temperature for 2 hours with fluorescence‐labeled secondary antibodies (Invitrogen). Primary antibodies included anti‐TNF‐α (Sigma‐Aldrich), anti‐Bax (Proteintech), anti‐TLR3 (Abcam), and anti‐TLR4 (Santa Cruz Biotechnology). To detect Bax levels, samples were treated with 10 mmol/L citrate buffer (pH 6.0) at 98°C for 20 minutes and then reacted with the antibody. In the control experiments, the primary antibodies were replaced by normal IgG (Abcam). After the secondary reaction, cells were washed with PBS and mounted on a cover glass for observation under a fluorescence microscope (Keyence). Image analysis was performed using Photoshop software (Adobe Systems Incorporated) to calculate the fluorescence intensity per cellular area.
9
Western Blot Analysis of Protein Markers
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The protein lysates were prepared with protease inhibitors cocktail‐contained radioimmunoprecipitation assay lysate (Beyotime). The protein samples were boiled at 98°C for 10 min. Next, 60 μg of proteins was used for analysis on 10% sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (SDS‐PAGE; Bio‐Rad, Hercules, CA). The SDS‐PAGE gels were transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes (Bio‐Rad) for 45 min. Then, the protein‐carried PVDF membranes were blocked with 2% bovine serum albumin, and they were incubated with the indicated primary antibody overnight. Afterward, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody for 45 min diluted at 1:50,000 (Bioworld Technology, Nanjing, China). Importantly, the primary antibodies including anti‐GAPDH, anti‐FBXW7, anti‐Bax, anti‐Bcl‐2, anti‐TNF‐α, anti‐IL‐1β, anti‐IL‐6, anti‐CAT, anti‐GSH, and anti‐SOD1 were purchased from Sigma‐Aldrich.
10
Inflammasome Activation in Lung Fibroblasts
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