Bronchoalveolar lavage fluid (BALF) was obtained by instilling 1 ml PBS through the tracheal cannula and suctioning back with a volume of 0.8–0.9 ml. The BALF was centrifuged at 12,000 g for 20 min at 4 °C as soon as finish the collection, and the supernatant was stored at −80 °C. The protein concentration was measured by the bicinchoninic acid (BCA) method using enhanced BCA protein assay kit (Beyotime, Shang Hai, China). The samples were separated using 10% SDS-PAGE and electro-transferred onto nitrocellulose membrane (Bio-Rad, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and were then incubated with primary antibodies in 5% BSA (in TBS + 0.1%Tween 20) overnight at 4 °C. The β-tublin (1:3000,Santa cruz, USA) was used as a loading control. The membranes were washed three times for 5 min each in TBS + 0.1%Tween 20, and were then incubated in the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa cruz, USA) for 2 h at room temperature. Finally, the protein bands were visualized using enhanced chemiluminescence (ECL). The relative quantity of proteins was analyzed using Image J and normalized to that of loading controls. Antibodies included: rabbit anti-TNF-α (1:1000, Abcam, USA), mouse anti-AQP4 (1:1000, Abcam, USA), rabbit anti -β-tublin (1:1000, Santa cruz, USA).
Rabbit anti tnf α
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Rabbit anti-TNF-α is a primary antibody that recognizes and binds to the tumor necrosis factor alpha (TNF-α) protein. TNF-α is a cytokine involved in inflammation and immune response. This antibody can be used for the detection and study of TNF-α in various applications, such as Western blotting, ELISA, and immunohistochemistry.
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Lab products found in correlation
Rabbit anti il 1β, by Abcam (14 mentions)
Mouse anti il 6, by Abcam (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (3 mentions)
Rabbit anti il 6, by Abcam (3 mentions)
Rabbit anti cox 2, by Abcam (2 mentions)
Rabbit anti mmp13, by Abcam (2 mentions)
Goat anti fstl1, by Abcam (2 mentions)
Mouse anti β actin, by Proteintech (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Rabbit anti p65, by Cell Signaling Technology (2 mentions)
Mouse anti cd68, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Rabbit anti il 4, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti insulin, by Cell Signaling Technology (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Rabbit polyclonal anti cyr61, by Thermo Fisher Scientific (2 mentions)
Donkey anti goat igg hrp, by Beyotime (2 mentions)
Goat anti mouse igg hrp, by Beyotime (2 mentions)
45 protocols using rabbit anti tnf α
1
Quantitative Protein Analysis of BALF
2
Antibody Panel for Immune Response Analysis
3
Western Blot Analysis of TNF-α and COX-2 in Skin Tissues
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Protein levels of TNF-α and COX-2 in the dorsal skin (n = 10 at each point in time) were analyzed according to our published procedure [17 (link)]. After sacrificing the animals, the tissues were homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′ tetraacetic acid (pH 8.0), 0.2% nonidet P-40, 10 mM ethylenediaminetetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium or thovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (DTT). The skin tissues were separated using 4‒20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, and the resolved proteins were transferred to a nitrocellulose membrane for 2 h at 40 V. Each membrane was incubated separately with the primary antibodies: rabbit anti-TNF-α (1:1000, Abcam) and rabbit anti-COX-2 (1:1000, Abcam) overnight at 4 °C. The membranes were washed with a washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween-20) and exposed to peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:1000 dilution at room temperature for 2 h. The membrane blots were developed using an enhanced luminol-based chemiluminescent (ECL) kit (Pierce Chemical, TX, USA).
4
Western Blot Analysis of Neuroinflammatory Proteins
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The protein was extracted using a total protein extraction kit (KeyGEN Biotech), and protein concentration was determined with the BCA Protein Assay Kit (Pierce Biotechnology). Total protein (20–50 μg) was separated by SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dried milk for 60 min and then incubated in the antibodies of rabbit anti-GABAARα1 (1:1000, Abcam), rabbit anti-GABAARα2 (1:1000, Abcam), rabbit anti-NKCC1 (1:400, Cell Signaling Technology), rabbit anti-KCC2 (1:8000, Abcam), rabbit anti-IL-1β (1:1000, Abcam), rabbit anti-TNF-α (1:1000, Abcam), mouse anti-TGF-β1 (1:200, Affinity) and mouse anti-β-actin antibody (1:5000, Abways Technology) as loading control. After thorough washing, the membranes were incubated with HRP-labeled secondary antibodies and developed using the ECL detection Kit (Millipore). Western blotting bands were scanned and analyzed using the Image J analysis software package (NIH). The densitometric values of the total protein were normalized by β-actin.
5
Immunohistochemical Analysis of Intervertebral Disc
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After being fixed with 4% paraformaldehyde, the nucleus pulposus tissues of both humans and rats were cut into 5-μm-thick slices for slide preparation. Tissues were incubated with theprimary antibodies as follows: goat anti-FSTL1 (1:200; Abcam plc, Cambridge, UK), rabbit anti- TNF-α, (1:300; Abcam), rabbit anti- MMP-13 (1:400; Abcam), rabbit anti- IL-1β, (1:200; Abcam). The slides after immunohistochemical staining were observed and analyzed by an electron microscope (Olympus, Tokyo, Japan). The results were quantified using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., USA). Each section was examined independently by two investigators in a blinded manner.
6
Western Blot Analysis of Inflammatory Markers
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The collected Raw264.7 cells and bone surrounding soft tissues were homogenized and lysed in lysis buffer including RIPA and phenylmethanesulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The lysates (30 μg/lane) were separated on 10% gels and were transferred onto polyvinylidene difluoride membranes (Beyotime Institute of Biotechnology). The membranes were blocked with 5% (w/v) BSA in Tris-buffered saline with 0.1% (w/v) Tween 20 (TBST) and probed with primary antibodies: mouse anti-ABCA1 (1: 1000; Abcam Biotechnology), rabbit anti-ABCG1 (1: 2500; Abcam Biotechnology), rabbit anti-Cav-1 (1: 1500; Abcam Biotechnology), rabbit anti-TNF-α (1: 1000; Abcam Biotechnology), rabbit anti-IL-6 (1: 1000; Cell Signaling Technology), rabbit anti-IL-1β (1: 1,000; Abcam Biotechnology), and mouse anti-β-actin (1: 4000; Proteintech, Rosemont, USA) at 4°C overnight. After washing, the bound antibodies were detected using optimal horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit IgG (1: 10 000) and goat anti-mouse IgG (1: 10 000; Merck Millipore, Darmstadt, Germany) and were visualized using the enhanced chemiluminescence reagents (Bio-Rad Laboratories, Hercules, USA). The relative levels of the target proteins to that of control β-actin were analyzed densitometrically using the Quantity One software (version 4.0, Bio-Rad).
7
Western Blot Analysis of Inflammatory Markers
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Western blot was performed as previously described.22 Total proteins (20 μg) were subjected to SDS‐PAGE and then transferred onto a polyvinyl indene difluoride membrane (PVDF; Millipore, Billerica, MA, USA). Nonspecific binding was blocked with PBST (0.5% Tween 20 in PBS) containing 5% non‐fat milk (Shandong Sparkjade Biotechnology Co., Ltd.) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with individual primary antibodies in PBST containing 1% non‐fat milk (mouse anti‐IL‐6, rabbit anti‐TNF‐α, rabbit anti‐IL‐1β, rabbit anti‐GFAP, 1:1000, Abcam; mouse anti‐GFAP, rabbit anti‐pSTAT3, mouse anti‐STAT3, rabbit anti‐JAK‐1, rabbit anti‐JAK‐2, 1:1000, Cell Signaling Technology, rabbit anti‐β‐actin, Biosynthesis Biotechnology Inc., Beijing, China). Following three washes with PBST, the membranes were then incubated with the secondary antibodies (HRP‐linked anti‐rabbit IgG; HRP‐linked anti‐mouse IgG; 1:2000; Cell Signaling Technology). Then, the proteins were detected by chemiluminescence reagents (Millipore) and observed using a ChemiDoc™ XRS+ Imaging System (Bio‐RAD, Hercules, USA). The protein levels were quantified by densitometry using Image J 1.4.3.67.
8
Western Blot Analysis of Neural Proteins
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Western blotting was performed as previously described [6 (link)]. Briefly, after brain protein sample preparation using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA), equal amounts of protein were loaded on an SDS-PAGE gel and run using electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked and incubated overnight at 4 °C with the following primary antibodies: goat anti-TREM2 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-PI3K (1:1000, Cell signaling, Danvers, MA, USA), rabbit anti-phosphorylated Akt (p-Akt, 1:1000, Cell signaling), rabbit anti-Akt (1:1000, Cell signaling), rabbit anti-TNF-α (1:1000, Abcam), rabbit anti-IL-1β (1:1000, Abcam), anti-Bcl-2 (1:2000, Abcam), anti-Bax (1:4000, Abcam), and goat anti-β-actin (1:5000, Santa Cruz Biotechnology). Appropriate secondary antibodies (1:3000, Santa Cruz; 1:5000, Abcam) were selected to incubate with the membrane for 2 h at room temperature. The bands were probed with an ECL Plus chemiluminescence regent Kit (Amersham Biosciences, Arlington Heights, PA, USA) and visualized with the image system (Versa Doc, model 4000, Bio-Rad, Hercules, CA, USA). Relative density of the protein immunoblot images were analyzed by ImageJ software (ImageJ 1.4, NIH, Bethesda, MD, USA).
9
Cell Culture Protocols with Antibody and miRNA
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All cell culture-related products were purchased from Gibco (Carlsbad, CA, USA). The following antibodies were used: goat anti-IL-6 and rabbit anti-IL-1β (Santa Cruz Biotechnology); rabbit anti-TNF-α (Abcam); rabbit anti-GAPDH (Cell Signaling Technology); all secondary antibodies were purchased from Santa Cruz Biotechnology. The miR-26b mimics (5′-UUCAAGUAAUUCAGGAUAGGU-3′ and 5′-CUAUCCUGAAUUACUUGAAUU-3′) and the negative controls (5′-UUCUUCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′) as well as the miR-26b inhibitor (5′-ACCUAUCCUGAAUUACUUGAA-3′) and the negative control (5′-CAGUACUUUGUGUAGUACAA-3′) were synthesized by Genepharma (Shanghai, China).
10
Immunohistochemical Analysis of Inflammatory Markers in Rat Lungs
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The lung tissues obtained from all three study groups of rats were steeped with buffered formalin in PBS, pH 7.2, and then infused in formalin at 4 °C overnight. After the fixation period, the lung tissues were embedded in paraffin. Six-micrometer sections of tissues were prepared, deparaffinized, and dehydrated. The samples were incubated in 50% formamide solution and 2 × standard sodium citrate buffer at 65 °C for 2 h and then incubated twice in 100 mM of sodium borate (pH = 8.5). The samples were blocked with 0.4% Triton X-100 in PBS and 10% goat serum for 30 min. Next, the sections were incubated with monoclonal primary antibodies in PBS containing 0.05% Tween-20 and 1% BSA at 4 °C overnight. The five used primary antibodies were against IL-1β (rabbit anti-IL-1β; 1:100, Abcam, Cambridge, UK), TNF-α (rabbit anti-TNF-α; 1:100, Abcam, Cambridge, UK), and VEGF (Rabbit anti-VEGF; 1:100, Abcam, Cambridge, UK). Afterward, the samples were washed with PBS and exposed to FITC-conjugated secondary antibodies (goat anti-rabbit; 1:200, Abcam, Cambridge, UK) diluted in the PBS at 37 °C for 90 min in the dark. DAPI was used for nuclear staining. The samples were visualized under a light microscope (Olympus, Japan) at 400 × magnification. FITC expression was measured by ImageJ software based on nuclear staining, indicating a positive reaction for the desired markers.
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