Mice under deep anesthesia were transcardially perfused with ice-cold PBS, followed by immersion in 4% paraformaldehyde. Coronal sections were prepared, blocked and then cut. Frozen sections were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-PDGFR-β (1:50, Cell Signaling Technology), mouse monoclonal anti-NG2 (a marker of cerebrovascular pericyte 1:100, Santa Cruz), rabbit polyclonal anti-TGF-β (1:100, Abcam), rabbit polyclonal anti-Ki-67 (1:100, Cell Signaling Technology), rabbit polyclonal anti-MCP-1 (1:200, Abcam) and rabbit polyclonal anti-TNF-α (1:50, Abcam). Then, the sections were incubated with appropriate host secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 594 or Alexa Fluor 637 (1:200, Invitrogen, USA). All sections were stained with 6-diamidino-2-phenylindole (DAPI, CA, USA) and photographed with a confocal microscope (TCS SP5, Leica).
Rabbit polyclonal anti tnf α
Manufactured by Abcam
Sourced in United States
Rabbit polyclonal anti-TNF-α is a laboratory reagent that can be used to detect the presence of tumor necrosis factor alpha (TNF-α) in various samples. It is a polyclonal antibody produced in rabbits that specifically binds to TNF-α.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Rabbit polyclonal anti tgf β, by Abcam (1 mentions)
Alexa fluor 637, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
Biotinylated goat anti rabbit, by Abcam (1 mentions)
Cryotome, by Thermo Fisher Scientific (1 mentions)
Antibody dilution buffer, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Alexa fluor 568 conjugated anti rabbit antibody, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti cxcr4, by Abcam (1 mentions)
Mouse monoclonal anti il6, by Santa Cruz Biotechnology (1 mentions)
Ecl plu, by Cytiva (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Rabbit anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Rabbit monoclonal anti smad3 phosphor c25a9, by Cell Signaling Technology (1 mentions)
5 protocols using rabbit polyclonal anti tnf α
1
Immunohistochemical Analysis of Cerebrovascular Pericytes
2
Immunohistochemical Analysis of Hepatic TNFα
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Prepared slides were deparaffinized as above. The presence of tumor necrosis factor alpha (TNFα) in the livers was determined using immunohistochemistry. Slides were submerged in pre-heated sodium citrate buffer (pH 6.0) and heated at 98°C for 20 minutes to retrieve antigens. Liver sections were then incubated with hydrogen peroxide, protein blocking, and streptavidin peroxidase solutions at ten minutes each. Following each incubation step, the sections underwent two washing cycles. Each cycle involved five minutes of gentle agitation in a solution of TBS (Tris-buffered saline, pH 7.0) with 0.025% Triton X-100. Liver sections were then incubated with primary and secondary antibodies i.e., rabbit polyclonal anti-TNFα (cat# ab6671, Abcam, USA) and biotinylated goat anti-rabbit (Abcam, USA). To visualize TNFα, DAB (3,3’-Diaminobenszidine) chromogen was used to produce a brown colored substance at the site of the primary antibody. For histopathology, slides were then counterstained with hematoxylin.
3
Immunofluorescent Localization of CXCR4 and TNF-α
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20 μm slices were cut by a cryotome (Thermo, USA). Slices were dried in ventilation for 30 minutes and then permeabilized by 0.25% triton-X 100 for 15 minutes. The sections were blocked with 10% bovine serum albumin (Sigma, USA) in phosphate buffer saline (PBS) diluent for 1 hour at room temperature. Then sections were incubated with rabbit monoclonal anti-CXCR4 (1 : 200, Abcam, USA) or rabbit polyclonal anti-TNF-α (1 : 200, Abcam) overnight at 4°C. After slices were washed with PBS for 10 minutes 3 times, added Alexa Fluor 568-conjugated anti-rabbit antibody (1 : 500, Abcam, USA) diluted in antibody dilution buffer (Dako, Denmark) for 1 h at room temperature and followed by nuclear staining using DAPI (Cell Signaling Technology, USA). Images were detected by a confocal microscope LSM 710 (Zeiss) and measured using ImageJ Software (National Institutes of Health, USA).
4
Quantification of Neuroinflammatory Markers
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Western blotting was carried out as previously described (Hu et al., 2011 (link); Liu et al., 2014 (link)). Briefly, brains were divided into left and right cerebral hemispheres, cerebellum, and brainstem. The cerebral hemispheres opposite the blood clots were homogenized and equal amounts of protein (50 μg) were loaded on a Tris-glycine gel, separated by electrophoresis, and transferred to a nitrocellulose membrane that was blocked with blocking solution followed by overnight incubation at 4°C with the following primary antibodies: rabbit polyclonal anti-TTP (1:200), mouse polyclonal anti-PP2A (1:500), rabbit polyclonal anti-TNF-α (1:1,000), and rabbit polyclonal anti-IL-8 (1:1,000) (all from Abcam): mouse monoclonal anti-IL-6 (1:200; Santa Cruz Biotechnology); rabbit polyclonal anti-B cell lymphoma (Bcl)-2 (1:500; Sigma–Aldrich); and mouse monoclonal anti-caspase-3 (1:500; Millipore, Temecula, CA, USA). Membranes were incubated with appropriate secondary antibodies (1:2,000; Santa Cruz Biotechnology) for 1 h at 21°C, and bands were detected with a chemiluminescence reagent kit (ECL Plus; Amersham Bioscience, Arlington Heights, IL, USA). Band intensity was quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA). β-actin (1:2,000; Santa Cruz Biotechnology) was used as a loading control.
5
Protein Expression Analysis in Heart and Lung
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Total protein was obtained from heart and lung tissues by sonication, centrifugation, and heat denaturation. The protein lysates were electrophoresed and separated on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto nitrocellulose membranes (Millipore, Inc, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies, including rabbit polyclonal Anti-Collagen I (1:800, ABcam, Inc.), mouse monoclonal anti-TGF-β1 (1:500, Abcam, Inc.), rabbit monoclonal anti-Smad3 (phosphor C25A9) (1:500, Cell Signaling Technology, Inc.), rabbit polyclonal anti-TNF-α (1:1000, Abcam, Inc.), mouse monoclonal anti-TLR4 (1:2000, Abcam, Inc.), and rabbit anti-NF-κB p65 (1:800; Santa Cruz, Inc.). The membranes were then incubated with the secondary antibody (1:2000) at room temperature for 2 h. Electrochemiluminescence was induced and the Gene Gnome Gel Imaging System (Syngene Co.) was used to capture the resulting images. Image J (NIH image, Bethesda, MD) was used to analyze the gel images. The results were expressed as density values normalized to GAPDH.
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