For immunocytochemistry, colonic tissues were steeped with 5% paraformaldehyde and then were paraffined and cut into 4 μm thick. Then anti-TLR4 antibody and anti-NF-κB antibody (1 : 200, Abcam, Cambridge, UK) were used in this experiment. Nonimmunized mouse IgG at equal concentrations was used as a negative control for each primary antibody. Rabbit anti-rabbit IgG and goat anti-mouse IgG (1 : 200, ZSGB-BIO, Peking, China) were used as the secondary antibodies. Images were analyzed using Image Pro Plus 6.0 software (Media Cybernetics UK, Marlow, UK). The immunological histological chemistry (IHC) indexes for TLR4 and NF-κB were determined as average integral optical densities according to the following equation: AIOD = (positive area/total area) × IOD.
Anti nf κb antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-NF-κB antibody is a laboratory reagent used to detect and quantify the presence of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in biological samples. NF-κB is a transcription factor that plays a crucial role in the regulation of various cellular processes, including immune response, inflammation, and cell survival.
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Lab products found in correlation
Anti tlr4 antibody, by Abcam (2 mentions)
Anti tnf α antibody, by Abcam (2 mentions)
Anti p nf κb antibody, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti myd88, by Abcam (1 mentions)
Anti tlr2, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Pierce ecl western blotting substrate, by Abcam (1 mentions)
Fitc conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Diamidino phenyl indole dapi, by Vector Laboratories (1 mentions)
Anti il 1β antibody, by Abcam (1 mentions)
Anti cox 2 antibody, by Abcam (1 mentions)
Anti il 6 antibody, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Abcam (1 mentions)
Hrp conjugated goat anti mouse antibody, by Abcam (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Anti bax antibody, by Abcam (1 mentions)
17 protocols using anti nf κb antibody
1
Immunocytochemistry of Colonic TLR4 and NF-κB
2
Immunohistochemical Analysis of NF-κB
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Before immunohistochemistry analysis, all slides were air-dried. The sections were fixed in four percent of paraformaldehyde for 10 min, washed, and permeabilised in PBS buffer for 20 min. The sections were blocked in 10% horse serum and 5% BSA for one hour. Anti-NF-κB antibody (Catalogue number ab36104, Abcam, Cambridge, MA, USA) was applied to the sections for 1 h at room temperature. The stain was performed by using hematoxylin stain as background color and 3,3-diaminobenzidine (DAB) stain to reveal positively stained tissue areas according to an earlier report [37 ].
3
Western Blot Analysis of Cardiac Proteins
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Cardiac tissues were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Then, 20 ug of protein was separated via 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were first blocked with 5% non-fat milk for 1 h at RT, and then incubated with specific primary antibodies overnight at 4°C, including anti-TLR4 (1:1,000; cat. no. ab13556; Abcam), anti-TLR2 (1:2,000; cat. no. sc-10739; Santa Cruz Biotechnology, Inc.), anti-RP105 (1:1,000; cat. no. Sc-13592; Santa Cruz Biotechnology, Inc.), anti-Bcl-2 (1:1,000; cat. no. ab196495; Abcam), anti-cleaved caspase-3 (1:1,000; cat. no. ab2302; Abcam), anti-MYD88 (1:1,000; cat. no. ab2064; Abcam) and anti-NF-κB antibody (1:1,000; cat. no. Ab32536; Abcam). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. nos. Ab97046 and Ab97057; Abcam) at RT for 1 h. The bands were exposed with Pierce ECL western blotting substrate (cat. no. 32106; Thermo Fisher Scientific, Inc). The expression of GAPDH (1:3,000; cat. no. Ab181602; Abcam) was detected to normalize the signals. The expression of proteins was qualified using ImageJ software (1.47; National Institutes of Health)
4
NF-κB Activation in Dorsal Root Ganglia Neurons
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Dorsal root ganglia neurons were seeded on poly-L-lysine coated glass and grown in 24-well plates. After drug treatment, neurons were fixed with 4% paraformaldehyde solution and stained with anti-NF-κB antibody (1:2,000, Abcam, United States) at 4°C overnight, followed by incubation with FITC-conjugated goat anti-mouse IgG (1:200, Jackson, United States) for 1 h at RT in the dark. Nuclei were stained for 5 min with diamidino-phenyl-indole (DAPI, Vector Laboratories). Neurons were visualized using a fluorescence microscope (IX71, Olympus, Japan).
5
Neuroinflammatory Markers in LPS-Induced Rat Hippocampus
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Twenty-six days after LPS induction, IL-1β, IL-6, TNF-α, cyclooxygenase-2 (COX-2), and NF-κB concentrations in the hippocampus were assayed according to a previously described procedure (Lee et al. 2014 (link)). Three rats from each group were deeply anesthetized via isoflurane inhalation (1.2%), and were sacrificed one day after behavioral testing. The IL-1β, IL-6, TNF-α, COX-2, and NF-κB concentrations were evaluated with competitive enzyme-linked immunoassays (ELISAs) using anti-IL-1β antibody (Abcam, Cambridge, MA, USA), anti-IL-6 antibody (Abcam), anti-TNF-α antibody (Abcam), anti-COX-2 antibody (Abcam), and anti-NF-κB antibody (Abcam) according to the manufacturer's protocols.
6
Antibody-based Apoptosis and Inflammatory Signaling Analysis
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The following materials were used: anti-Bcl-2 antibody (Abcam, 182,858), anti-Bax antibody (Abcam, 32,503), anti-GAPDH antibody (Abcam, 8245), anti-cleaved caspase-3 antibody (Cell Signaling Technology, 9664), anti-IL-1β antibody (Cell Signaling Technology,12,242), anti-IL-6 antibody (Cell Signaling Technology, 12,912), anti-IL-10 antibody (Abcam, 189,392), anti-TNF-α antibody (Abcam, 183,218), anti-TLR4 antibody (Abcam, 22,048), anti-MyD88 antibody (Abcam, 133,739), anti-NF-κB antibody (Abcam, 16,502), anti-p-NF-κB antibody (Abcam, 76,302), goat anti-rabbit HRP-conjugated antibody (Abcam 6721), and goat anti-mouse HRP-conjugated antibody (Abcam 6789).
7
Evaluation of Cell Injury Signaling
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Soluble dsDNAs in the supernatant were evaluated by dsDNA Assay kit (see Supplementary Materials ) [35 (link)]. Cell injury and the associated signal were evaluated by TUNEL assay [35 (link)] and immunostaining of NF-κB p65 subunit and STAT3 using anti-NFκB antibody (Abcam, Cambridge, UK) and anti-STAT3 antibody (Thermo Fisher Scientific), respectively, after fixation with 4% paraformaldehyde. The positive areas of TUNEL staining and immunostaining were measured using the ImageJ software as described [38 (link)]. The data were expressed as the mean ± standard error of the mean (SEM) from three independent experiments.
8
Hippocampal Protein Signaling Analysis
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Hippocampal tissues were homogenized with NP40 buffer containing 1% protease inhibitors and 1% phosphatase inhibitor (Sigma-Aldrich Co., MO, United States), followed by centrifugation at 12,000 g for 20 min at 4°C. The supernatant of the hippocampal homogenate was then collected. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (CWBio, China). Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to the PVDF membrane (Bio-Rad, CA, United States). The membranes were blocked for 1 h with 5% non-fat milk, followed by incubation with the primary antibody of anti-RIP1 (1:200, Cell signalling technology, Danvers, United States) and either RIP3, MLKL, or anti-NF-κB antibody (1:1500, Abcam, MA, United States), respectively, followed by incubation for another hour with the IRDye®800CW goat anti-rabbit secondary antibody (1:8000, 926-32211, LI-COR®, United States). Immunoblotting bands were visualized under Odyssey CLx infrared imaging systems (LI-COR®, United States). Protein levels were quantified by densitometry using Image J software (National Institutes of Health, MD, United States) and were normalized to GAPDH, respectively.
9
Immunofluorescence Staining of DRG Neurons
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DRG tissues and primary DRG neurons were fixed with 4% paraformaldehyde for 20 min and washed with phosphate-buffered saline (PBS). The sections were immersed in 0.4% Triton X-100 for 15 min and blocked in 5% BSA for 1 h at room temperature. The sections were then stained with βⅢ-tubulin mouse monoclonal antibody (1:500, Abcam), anti-TRPV1 (VR1-C)-mouse specific antibody (1:300, Neuromics, INA), and anti-NF-κB antibody (1:500, Abcam, United States) at 4°C overnight, followed by incubation with FITC-conjugated goat anti-mouse IgG (1:200, Proteintech, CHN) for 1 h at room temperature. Sections were treated with DAPI, and immunofluorescence images were visualised using a fluorescence microscope (IX71, Olympus, Japan).
10
Investigating NF-κB Modulation by Corydaline Alkaloids
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The cultured macrophage cells were placed on glass coverslips and treated with dehydrocorydaline, corydaline, and corydalmine at concentrations of 20, 60, and 90 µM, respectively. In parallel, the positive control cells (treated with dexamethasone and LPS) and negative control cells (not treated with drug and LPS) were performed. After being washed with PBS, the cells were fixed with 4% formaldehyde for 15 min. Then, the cells were blocked by 0.5% BSA with 0.2% Triton X-100 for 1 hour at room temperature. The cells were incubated with anti-NF-κB antibody (Abcam) at 1 : 500 for overnight at 4°C, followed by the Alexa Fluor 555-conjugated anti-rabbit antibody (Sigma) together with 4′,6-diamidino-2-phenylindole (DAPI) staining (5 mg/mL) for 2 hours at room temperature (Sigma). After being washed, the samples were then examined by a Leica SP8 confocal microscope.
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