Infectivity of CD8+ T cells was assessed via surface staining with anti-CD3ε APC, anti-CD8α FITC, and anti-Thyl.1 PE. Biolegend antibodies used in experiments anti-CD3ε PE/Cy7, anti-CD8α APC, anti-CD16/32, anti-CD27 PE, anti-CD44 PE, anti-CD45 PerCP/Cy5.5, anti-CD62L PE, anti-CD69 PE, anti-CD107a PE, anti-CD279 PE, anti-IFNγ APC, anti-Granzyme B PE, anti-Lag3 PE, anti-SIINFEKL:H-2Kb PE, anti-Thy1.1 PE, and anti-Tim3 PE. Anti-human/mouse DHX37/Dhx37 purchased from Novus Biologicals and anti-NF-κB p65 purchased from Invitrogen were used for immunoblotting. Anti-human DHX37 and anti-human PDCD11 purchased from Bethyl Laboratory was used for immunoprecipitation. Anti-β-actin purchased from Invitrogen and anti-GAPDH purchased from Santa Cruz Biotechnology was used for loading control. For surface stains, cells were stained on ice for 30 mins. Samples were collected on a BD FACSAria cell sorter with 3 lasers, and analyzed using FlowJo software 9.9.6 (Treestar, Ashland, OR) on a MAC® workstation.
Anti nf κb p65
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-NF-κB p65 is a primary antibody that binds to the p65 subunit of the NF-κB transcription factor. NF-κB p65 plays a key role in the regulation of immune and inflammatory responses. This antibody can be used for various applications, including Western blotting, immunohistochemistry, and immunocytochemistry.
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Lab products found in correlation
Anti caspase 3, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Mowiol 40 88, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Nis elements software, by Nikon (1 mentions)
C1 confocal microscope, by Nikon (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Solarbio (1 mentions)
Anti hsp90, by Proteintech (1 mentions)
Ecl plus detection system, by Merck Group (1 mentions)
Anti p akt, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Elisa kits for tumor necrosis factor α tnf α, by R&D Systems (1 mentions)
Ho 1 elisa kit, by MyBioSource (1 mentions)
Il 1β, by R&D Systems (1 mentions)
7 protocols using anti nf κb p65
1
Multiparameter Analysis of CD8+ T Cell Responses
2
NF-κB Translocation Assay in Prostate Cells
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To perform the NF-κB translocation assay, BPH-1 and PC3 cells were firstly seeded on glass coverslips in 24-well plates and cultured as previously indicated. When cells reached approximately 30% confluence, they were fixed with 4% formaldehyde (Sigma–Aldrich, Merck, Darmstadt, Germany), permeabilized with 0.1% Triton X-100 (Sigma–Aldrich, Merck, Darmstadt, Germany) in PBS, and stained 1 h with rabbit monoclonal anti-NF-κB p65 (Invitrogen, Life Technologies, Milan, Italy). After PBS washes, they were incubated with anti-rabbit secondary antibody Alexa Fluor 488 (Molecular Probes, Invitrogen, Milan, Italy) for 1 h at room temperature. Cells were then washed with PBS and stained with Hoechst (1:10,000) (Invitrogen, Life Technologies, Milan, Italy). The coverslips were finally mounted on glass slides by using Mowiol 40–88 (Sigma–Aldrich, Merck, Darmstadt, Germany). Images were acquired through a 60x CFI Plan Apochromat Nikon objective with a Nikon C1 confocal microscope and finally analysed using NIS Elements software (Nikon Instruments, Florence, Italy), NIH Image J version 1.52t, and Adobe Photoshop CS4 version 11.0.2 (Adobe, San Jose, CA, USA).
3
Protein Expression Analysis in Myocardial Tissues
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Myocardial tissues from each group were homogenized using radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China) with an ultrasonic grinder, and then placed on ice for 30 min to dissolve the tissues. Subsequently, a 2 mL centrifuge tube was centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was collected and the protein concentration was determined using an enhanced BCA protein assay kit (Beyotime, Shanghai, China). Then, 10 μL of protein per sample were loaded and separated by 10% SDS-PAGE and then transferred onto the PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA for 1 h at 25°C, and then incubated with the primary antibodies (anti-HSP90, 1:5,000, Proteintech, Chicago, USA; anti-p-Akt, 1:5,000, Proteintech; anti-β-actin, 1:5,000, Proteintech; anti-C5a, 1:5,000, Invitrogen; anti- NF-κB p65, 1:1,000, Invitrogen) at 4°C for 24 h. Then the membranes were thoroughly washed using TBST and incubated with the secondary antibody, an HRP-labeled goat anti-rabbit IgG (1:12,000) at 25°C for 1 h. The ECL Plus detection system (Millipore Corporation, Billerica, MA, USA) was used for the detection of band immune-detection according to the manufacturer’s instructions.
4
Comprehensive Cardiac Biomarker Evaluation
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The ISO was procured from Sigma (St. Louis, MO, USA). The TAX was purchased from Biosynth Carbosynth (Berkshire, UK). A Cardiac troponin I (cTnI) ELISA kit was obtained from Kamiya (Tukwila, WA, USA). Kits for creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) activities were procured from Spinreact (Girona, Spain). A HO-1 ELISA kit was obtained from MyBioSource (San Diego, CA, USA). The ELISA kits for tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were supplied by R&D Systems (Minneapolis, MN, USA). Anti-NF-κB p65, anti-caspase-3, and anti-Nrf2 antibodies were obtained from ThermoFisher (Waltham, MA, USA; 1:100 dilution), while anti-Bax and anti-Bcl-2 antibodies were supplied by Abcam (Cambridge, MA, USA; 1:100 dilution).
5
Anti-inflammatory Mechanism of Compounds
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Lipopolysaccharide (LPS), carrageenan, 4,5-diCQA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphoric acid, sulfanilamide, and N-(1-naphthyl)ethylenediamine dihydrochloride were purchased from Sigma–Aldrich (St. Louis, MO, USA). A prostaglandin E2 enzyme-linked immunosorbent assay (PGE2 ELISA) kit was purchased from R&D Systems (Minneapolis, MN, USA). All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), except for anti-inducible NO synthase (iNOS) (Abcam, Cambridge, UK), anti-NF-κB(p65), and anti-α-tubulin (Thermo Fisher Scientific, Waltham, MA, USA). Dulbecco’s modified Eagle’s medium and penicillin/streptomycin solution were purchased from WelGene (Daegu, Korea). Fetal bovine serum was purchased from ATLAS Biologicals (Fort Collins, CO, USA).
6
Doxorubicin-Induced Oxidative Stress Modulation
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DOX was purchased as Adriablastine (50 mg doxorubicin hydrochloride, Pharmacia & Upjohn, Milan, Italy). Chrysin, GSH, Ellman’s reagent [5,5-dithio-bis (2-nitrobenzoic acid); DTNB], bovine serum albumin, dimethylsulphoxide (DMSO) and thiobarbituric acid were purchased from Sigma Chemical Co. (St Louis, MO, USA). Anti total p38, anti phospho p38, anti total JNK and anti phospho JNK antibodies were purchased from R & D systems (Minneapolis, USA). Anti total AKT and anti phospho AKT antibodies were purchased from Biovision Co. (San Francisco, USA). Anti GAPDH antibody was purchased from Santa cruz biotechnology (Santa cruz, Texas, USA). Anti NF-κB p65, VEGF, p53, cytochrome c, caspase 3 antibodies were purchased from Thermo Fisher Scientific Co. (Waltham, MA, USA). All other chemicals were of the highest purity grade commercially available.
7
Immunohistochemical Analysis of Oxidative Stress Markers
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For IHC, the deparaffinized and hydrated sections were treated with 0.05 M citrate buffer (pH 6.8) for antigen retrieval followed by 0.3% hydrogen peroxide. The nonspecific antigen-antibody binding was blocked through the addition of normal serum for 20 min. The sections were washed in PBS and probed overnight at 4 °C with anti-NF-κB p65 (ThermoFisher, Waltham, MA, USA), anti-Bax (Abcam, Cambridge, MA, USA), anti-Bcl-2 (Abcam, Cambridge, MA, USA), anti-caspase-3 (ThermoFisher, Waltham, MA, USA), and anti-Nrf2 (ThermoFisher, Waltham, MA, USA). After washing in PBS, anti-mouse secondary antibodies were added to the slides, and DAB was used for color development. Then, the sections were counterstained with Mayer’s hematoxylin, and examined under a light microscope. The staining labelling indices of the caspase-3 and NF-κB p65 were presented as a percentage equivalent field of positive control expression. The immunostaining intensity of anti-Bcl-2 and anti-Nrf2 antibodies was determined through a percent of the positive area using image J analysis software (NIH, Bethesda, MD, USA).
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