Rat tissues were perfused with phosphate-buffered saline (PBS), sliced, and fixed with 4% paraformaldehyde for the immunohistochemical experiments. Intestinal samples were incubated overnight in PBS with 6.8% sucrose, dehydrated with acetone, and paraffin-embedded. Before staining, semi-thin sections were incubated for 5 min at 37°C in 0.01% trypsin/0.1% CaCl2 (pH 7.8). Sections were incubated for 5 h at 37°C with NF-κB antibodies (Abcam, UK), and then sections were again rinsed three times in 0.1 M PBS for 5 min each, incubated with secondary antibody for 15–20 min, rinsed three times in 0.1 M PBS for 5 min each, incubated at 37°C for 30 min, and rinsed four times in 0.1 M PBS for 5 min each. Diaminobenzidine (DAB) was applied for 3–5 min until a brown-colored reaction product was observed. After rinsing the sections in distilled water three times, the sections were counterstained with hematoxylin for 10 s, rinsed three times in distilled water, and dehydrated with an ascending concentration (i.e. 80%, 90%, 95% and 100%) of ethanol for 10 min each. Sections were then cleaned and mounted and coverslips were added for immunohistochemical study. Images were captured with a Nikon photo microscope equipped with a digital camera.
Nf κb antibody
Manufactured by Abcam
Sourced in United States
The NF-κB antibody is a tool used in research applications to detect and quantify the presence of the NF-κB protein. NF-κB is a transcription factor that plays a crucial role in regulating immune responses, inflammation, and cell survival. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), to study the expression and localization of NF-κB in biological samples.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Photomicroscope, by Nikon (1 mentions)
Thermo scientific rip kit, by Thermo Fisher Scientific (1 mentions)
Dna extraction kit, by Bio-Rad (1 mentions)
Chip assay kit, by Beyotime (1 mentions)
Pierce agarose chip kit, by Epigentek (1 mentions)
F4 80 antibody, by Abcam (1 mentions)
Lps escherichia coli 0111 b4, by Merck Group (1 mentions)
4 6 diamino 2 phenylindole dapi, by Abcam (1 mentions)
Myeloperoxidase mpo antibody, by Abcam (1 mentions)
P nf κb antibody, by Abcam (1 mentions)
Tlr4 antibody, by Cell Signaling Technology (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Pyrrolidine dithiocarbamate, by Merck Group (1 mentions)
Gapdh antibody, by ZSGB-BIO (1 mentions)
Claudin 5 antibody, by Abcam (1 mentions)
Gsk3β antibody, by Abcam (1 mentions)
Ultrafiltration centrifuge tube, by Merck Group (1 mentions)
Tnf α antibody, by Abcam (1 mentions)
12 protocols using nf κb antibody
1
NF-κB Immunohistochemistry in Rat Tissues
2
RIP Assay for LOC645166-NF-κB Interaction
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A RIP assays were carried out to study whether LOC645166 could interact with NF-κB using a Thermo Scientific RIP kit (Thermo, Waltham, MA, USA) according to the manufacturer’s instructions. NF-κB antibodies were purchased from Abcam and Rabbit IgG (Abcam) was used as the negative control. Purified RNA was subjected to RT-qPCR analysis. The sequences of each primer were shown in supplementary table 5.
3
ChIP Assay of miR-518a-5p Promoter
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A ChIP assay kit (#P2078, Beyotime, Shanghai, China) was used according to the manufacturer's protocols. HTR8/SVneo cells were cross-linked with 1% formaldehyde for 12 min and sonicated into DNA fragments of 200 and 1000 bp. Cell lysates were incubated with the NF-κB antibodies (Abcam) that were coated with protein A/G beads at 4°C overnight. The goat-anti-rabbit IgG served as a negative control. The immunocomplexes that were bound to protein A/G beads were then eluted with elution buffer to remove the nonspecific binding. Samples were treated with 5 M NaCl and heated at 65°C overnight to eliminate histone-DNA crosslinks. Next, proteinase K was added followed by incubation at 45°C for 2 h. A DNA Extraction Kit (BIO-RAD) was used to purify the bound DNA fragments. Products were finally analyzed by real-time PCR using the primers specific to miR-518a-5p promoter.
4
Uncovering Wisp2-Mediated NF-kB Regulation
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To assess the influence of Wisp2 overexpression on the interaction NF-κB and target genes, ChIP test was carried out with Pierce Agarose Chip Kit (EpiGentek Group, Inc., Farmingdale, NY, USA). Cells were transfected with vector or pcDNA-Wisp2 and precipitated with NF-κB antibody (Abcam). Non-precipitated genomic DNA input was amplified as an input control. Normal rabbit IgG was applied for the negative control. After purification, the concentrations of DNAs were assessed.
5
Anti-inflammatory effects of STV-Na
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STV‐Na was provided by Key‐Pharma Biological Inc. LPS (Escherichia coli 0111: B4) was retrieved from Sigma Chemical Co. myeloperoxidase (MPO) antibody, F480 antibody, GAPDH antibody, NF‐κB antibody, p‐NF‐κB antibody and 4,6‐diamino‐2‐phenyl indole (DAPI) were obtained from Abcam. TLR4 antibody was obtained from Cell Signaling Technology. Inc. ROS fluorescent probes were purchased from Bestbio Biologicals Ltd. The anti‐rabbit immunoglobin G (IgG) antibody was purchased from Zhongshan Jinqiao Biological Co. ExCell Bio Co was the supplier of all enzyme‐linked immunosorbent assay (ELISA) kits. Wright–Giemsa staining solution, hematoxylin‐eosin (HE) Staining Kit, bicinchoninic acid (BCA) Protein Assay Kit, 5% bull serum albumin (BSA) Blocking Buffer, goat serum and Tris buffered saline containing 0.1%Tween 20 (TBST) were acquired from Solaibao Life Sciences Co. (Beijing, China).
6
Investigating NF-κB Pathway Modulation
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Pyrrolidine dithiocarbamate (PDTC) was purchased from Sigma-Aldrich. TLR4 antibody was obtained from Bioss (USA), NF-κB antibody was bought from Abcam (USA), and GAPDH antibody was from ZSGB-BIO (China).
7
Assessing Hepatic and Renal Apoptosis and Inflammation
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Caspase-3 and nuclear factor Kappa B protein (NF-κB) expression were examined for the determination of both apoptosis and inflammation within the hepatic and renal tissue sections. Briefly, formalin-fixed paraffin-embedded tissue sections were deparaffinized, microwaved and then incubated with caspase-3 antibody or NF-κB antibody (Abcam, Ltd., USA) at 1/200 dilutions overnight at 4°C, then washed and incubated with Peroxidase Block (Sakura BIO) and reagent required for the detection of the antigen-antibody complex (Power-Stain 1.0 Poly HRP DAP Kit, Sakura, REF. 52–0017). The sections were treated with DAB chromogen substrate for 10 mins and then the slides were counterstained by Hematoxylin, examined under a light microscope and analyzed using Image J software to evaluate mean % area of caspase-3 protein expressions in different groups. In the case of NF-κB immunostaining, positive immunostaining nuclei were blindly counted in 5 random microscopic fields per 3 random sections per group at × 400 magnification.
8
Immunohistochemical Analysis of TRIM23 and NF-κB
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Formalin‐fixed paraffin‐embedded tissues were sliced consecutively into 4 µm sections, and then subjected to immunohistochemistry analysis. The sections were incubated with TRIM23 (Abcam; 1:400 dilution) and NFκB antibody (Abcam; 1:400 dilution) at 4°C overnight. After washing in PBS, sections were further incubated with HRP‐conjugated secondary antibody for 30 minutes at 37°C. Then, substrate‐chromogen (DAB) solution was used to incubate the tumor tissues for 10 minutes. Finally, automated hematoxylin was used to counterstain the slides for 5 minutes. The immunostaining was microscopically evaluated by two independent pathologists. A semiquantitative scoring system was based on the staining intensity and proportion of positive cells.14
9
Neuroprotective Lipid Nanoparticles for Parkinson's
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1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and lecithin (Lec) was purchased from Aladdin chemical reagent company (Shanghai, China). l -α-lysophosphatidylcholine (Lpc), cholesterol and stearic acid (SA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Borneol was purchased from Selleck Chemicals (Shanghai, China). Exenatide (Exe) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Dir, Did, and DiI were purchased from Yeasen Co. Ltd (Shanghai, China). Ultrafiltration centrifuge tube (MWCO = 100 kDa) was purchased from Merck Millipore (Darmstadt, Germany). Chitosan (CSO), degraded using chitosanase in our laboratory (Mw = 18 kDa), was utilized. Tyrosine hydroxylase (TH) antibodies were purchased from Cell Signaling Technology Co.Ltd (Danvers, MA, USA). The commercial enzyme-linked immunosorbent assay (ELISA) kits of dopamine and GDNF were purchased from Elabscience Biotechnology Co., Ltd (Wuhan, China). 1-Methyl-4-phenyl-1,2,3,6- tetrahydropyridine hydrochloride (MPTP) was purchased from Aladdin chemical reagent company (Shanghai, China) and used to induce PD model in C57BL/6 mice. α-synuclein antibody, GSK-3β antibody, NF-κB antibody, claudin-5 antibody and TNF-α antibodies were purchased from Abcam (Boston, USA).
10
Cardiac Tissue Protein Extraction and Analysis
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The total protein of mouse cardiac tissues and cells was extracted with the Pierce IP Lysis Buffer (Thermo Fisher Scientific Inc.). The concentration of proteins was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of extracted protein (60 μg) were separated by 10% SDS‐PAGE and were transferred to PVDF membranes. The membranes were blocked with 5% non‐fat milk for 2 hours and incubated with primary antibodies: Bcl‐2 antibody, Bax antibody and NF‐κB antibody (Abcam, Cambridge, MA, USA) at 4°C overnight and incubated with HRP‐conjugated secondary antibodies at room temperature for 1 hour. GAPDH antibody was used as the internal control. The bands were analysed using the Enhanced Chemiluminescence Kit (GE Healthcare, Chicago, IL, USA).
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