Berberine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Before being added to the medium, BBR was dissolved in DMSO at an appropriate volume. Fetal bovine serum was purchased from Gibco (Grand Island, NY, USA). Rabbit anti-mouse NF-κB p65, phospho-NF-κB p65, phospho-IκB and IκBα, and β-actin antibodies were purchased from Santa Cruz Biotechnology (CA, USA). R700-labeled anti-CD3, BV786-labeled anti-CD4, BB515-labeled anti-CD25, BV421-labeled anti-Foxp3, and BV650-labeled anti-IL-17A antibodies were provided by Becton, Dickinson, and Company (Franklin Lakes, USA). The antibodies against CD25 were purchased from R&D Systems (Minneapolis, MN, USA). The enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biosource (Worcester, MA, USA) for measuring the contents of murine IL-1β, IL-6, IL-10, and IL-17A.
Rabbit anti mouse nf κb p65
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit anti-mouse NF-κB p65 is a primary antibody that recognizes the p65 subunit of the NF-κB transcription factor in mouse samples. It can be used for Western blotting, immunohistochemistry, and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phospho nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Berberine, by Merck Group (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fitc conjugated anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Fv1000, by Olympus (1 mentions)
Horse serum, by Merck Group (1 mentions)
Rat anti mouse cd68, by Bio-Rad (1 mentions)
Rat anti mouse il 17a, by R&D Systems (1 mentions)
Rabbit anti mouse cd4, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
4 protocols using rabbit anti mouse nf κb p65
1
Berberine Modulates Immune Signaling
2
Immunofluorescence analysis of NF-κB p65 activation
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Immunofluorescence analysis was performed as described previously [25 (link)]. Briefly, BMDMs were infected with Mmass after they were fixed with 4 % paraformaldehyde (for 10 min) and 0.25 % Triton X-100 (for 15 min) at RT. The cells were stained with primary antibody (rabbit anti-mouse NF-κB p65, 1:400, for 2 h; Santa Cruz) and secondary antibody (anti-rabbit AlexaFluor 488, 1:400, for 1 h; Invitrogen) at RT. Nuclei were stained with 1 μg/mL DAPI (Sigma-Aldrich). The slides were imaged using a Zeiss LSM510META confocal microscope (Zeiss, Germany).
3
Immunofluorescence analysis of NF-κB in B. abortus-infected macrophages
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RAW 264.7 macrophages at 106 cells per well were infected with B. abortus and incubated for 24 h at 37 °C in 5% CO2. Cells were then fixed with 4% paraformaldehyde, incubated at 37 °C for 1 h, permeabilized with 0.1% Triton X-100 for 10 s at 4 °C and incubated with blocking buffer (2% goat serum in PBS) for 1 h. Rabbit anti-mouse NF- κB p65 and FITC-conjugated anti-rabbit IgG were subsequently added as primary and secondary antibodies (Santa Cruz), respectively. The cells were mounted with Permafluor mounting medium and analyzed by using a laser scanning confocal microscope (Olympus FV1000, Japan). The images were processed using FV10-ASW Viewer 3.1 software.
4
Immunohistochemical Analysis of Aortic Valve
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The extent of aortic valve was assessed by immunohistochemistry. Lipid depositions were assessed following Oil Red O staining and nuclei were stained with hematoxylin. CD4 + T cells, valvular interstitial cells (VICs) and macrophages were stained in aortic valves. Briefly, after blocking by horse serum (Sigma Aldrich, USA), samples were reacted with rabbit antimouse CD4 (1:500 dilution, Abcam, ab183685, UK), FITC-conjugated mouse anti-mouse smooth muscle cell α-actin (αSMA, 1:500, Sigma Aldrich, F3777, USA), rat anti-mouse CD68 (1:100 dilution, Serotec, MCA1957, UK), rat anti-mouse IL17A (1:100 dilution, R&D, MAB721, USA), goat anti-mouse IL17RA (1:100 dilution, R&D, AF448, USA) and rabbit anti-mouse NF-κB (p65) (1:100 dilution, Santa Cruz, sc-372, USA) antibodies at 4°C overnight in the dark, respectively. After incubation with secondary antibody for 1 h at room temperature in the dark, nuclei were counterstained with DAPI (Vector Laboratories, USA). Images were recorded and analyzed with Image J software.
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