Heat-induced epitope retrieval was performed on dewaxed slides in citrate buffer (pH 6.0) at 95 °C for 40 min. Then, sections were exposed to peroxidase blocking solution (3% H2O2) prior to the addition of the primary antibody, anti-NF-κB p65 antibody (Abcam, Cambridge, MA, USA) diluted to 1:200 in PBS. After incubation with the primary antibody overnight at 4 °C, the slides were washed three times with PBS, and incubated with horseradish peroxidase (HRP)-incubated secondary antibody (Abcam, Cambridge, MA, USA) for 45 min. The sections were washed again with PBS three times. Subsequently, the slides were developed by diaminobenzidine (DAB) and counterstained with hematoxylin. Finally, the slides were observed under a light microscope.
Anti nf κb p65 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-NF-κB p65 antibody is a primary antibody that specifically recognizes the p65 subunit of the nuclear factor-kappa B (NF-κB) transcription factor. NF-κB is a key regulator of inflammatory and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti ki67 antibody, by Abcam (2 mentions)
Bx 60 system, by Adobe (2 mentions)
Anti myd88 antibody, by Abcam (2 mentions)
Anti tlr4 antibody, by Abcam (2 mentions)
Anti lgr5 antibody, by Abcam (1 mentions)
Epifluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti fade mounting solution, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence ecl detection system, by Cytiva (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Refine detection kit, by Leica (1 mentions)
Bond max, by Leica (1 mentions)
Anti bax antibody, by Abcam (1 mentions)
Anti nf κb p65 phospho s536 antibody, by Abcam (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
23 protocols using anti nf κb p65 antibody
1
Immunohistochemical Localization of NF-κB
2
Immunohistochemistry of Intestinal Markers
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Paraffin-embedded sections were dewaxed and rehydrated. The sections were then immersed in Tris/EDTA (pH 9.0) and incubated for 15 min at 98 °C for antigen retrieval. The sections were incubated with serum for 30 min at room temperature to block non-specific antigen-binding sites. Anti-Ki67 antibody, anti-NF-κB p65 antibody, anti-Lgr5 antibody (Abcam, Cambridge, MA, USA), anti-lysozyme antibody, and anti-Villin antibody (Proteintech, Chicago, IL, USA) was diluted as recommended by the manufacturer, added to the sections, and incubated overnight at 4 °C. Unbound antibodies were washed away with PBS. Sections were incubated in 0.3% H2O2 for 15 min to block endogenous peroxidase. Secondary antibody was added and incubated at 37 °C for 2 h. DAB was added to detect positive cells.
3
Immunofluorescence Analysis of NF-κB in Cardiac Tissue
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Paraffin-embedded left ventricle tissue sections (4 μm in thickness) were deparaffinized as described above for WGA staining. After blocking with 1% BSA for 1h at room temperature, slides were incubated with anti-NFκB P65 antibody (Abcam, Cat. No: ab16502) at 4°C overnight. The primary antibody was then washed with TBS-T solution for 3 times, followed by incubating with a secondary anti-rabbit antibody conjugated with Alexa 488 for 2h at room temperature. The slides were then incubated with mounting medium containing DAPI for nuclear staining and mounted with coverslip. Fluorescent signals were visualized using a Leica confocal microscope with a 63x oil lens. Five images were taken from each slide. Data from 4–5 animals in each group were analyzed by Two-Way ANOVA with GraphPad software version 7.0.
4
Measuring NF-κB Translocation by Immunofluorescence
5
Blueberry Extract Modulates Inflammatory Pathways
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Indometacin tablets were obtained from Linfen Qilin Pharmaceutical Co. Ltd. (Shanxi, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (CA, USA). LPS and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). The enzyme-linked immunosorbent assay (ELISA) transcriptase kits for TNF-α were purchased from CUSABIO (Wuhan, China). The primary antibodies anti-NF-κB p65 antibody and anti-P-NF-κB p65 antibody were purchased from Abcam (Cambridge Science Park, UK). Peroxidase-labeled rabbit anti-mouse, sheep anti-rabbit immunoglobulin, and an enhanced chemiluminescence (ECL) detection system were obtained from Amersham (Arlington Heights, IL, USA). All other chemicals were purchased from Sigma-Aldrich Chemical. The dried leaves of blueberry were purchased from Jiangsu, China.
6
Immunofluorescence Staining of Lung Tissue
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Tissue sections (6-8 μm) from frozen inflated lung tissue were first fixed in pre-chilled methanol for 10 minutes, washed with PBS followed by incubation for 1 hour at room temperature in 1% BSA in PBS. Tissue sections were then stained with unconjugated anti-NF-κB p65 antibody (Abcam, Cambridge, MA) (diluted 1:200 in 1% BSA in PBS), or anti-CD33 antibody () for 60 minutes, at room temperature. After PBS washes, sections were incubated with Alexa594 or Alexa 488 conjugated goat anti-rabbit secondary antibody, washed in PBS and stained with DAPI. Digital photomicrographs were acquired using an Olympus BX 60 system and processed with Adobe Photoshop software.
7
Immunohistochemical Analysis of Xenograft Tumors
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Subcutaneous xenograft tumors were prepared for IHC analysis. IHC was performed in a Leica Bond Max automated system (Leica Biosystems, Nussloch, Germany) using the Leica-Refine detection kit (Leica Biosystems, DS9800). Briefly, after deparaffinization, rehydration, and antigen retrieval, the sections were incubated with an anti-Ki-67 antibody (1 : 200, Abcam) and an anti-NF-κB p65 antibody (1 : 100, Abcam) for 30 min at room temperature. After washing, the sections were incubated with a horseradish peroxidase-conjugated secondary antibody, visualized with diaminobenzidine, and counterstained with hematoxylin. The stained slides were observed under a microscope (Olympus, Japan).
8
Western Blot Analysis of Apoptosis and NF-kB Signaling
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The total protein was extracted with the nucleoprotein and cytoplasmic protein extraction kit (Jiangsu Keygen Biotech Co., Ltd. China) and quantified by the BCA protein assay kit (Beijing Bomaide Gene Technology Co., Ltd., China). After that, the protein solution was subjected to electrophoresis, detached via SDS-PAGE (12% gels), and transferred to PVDF membranes. The PVDF membranes were blocked with 5% skim milk powder in TBST (Tris buffered saline with 0.5% Tween 20) for 2 h, and then incubated with primary antibodies overnight at 4° C. After washing with TBST for 5 times, the PVDF membranes were incubated with secondary antibody at room temperature for an hour. The protein bands were exposed by enhanced chemiluminescent (ECL) substrate kit (Labgic Technology Co., Ltd. Hefei, China) and analyzed by ImageJ software. The Anti-Bcl-2 antibody, Anti-Caspase-3 antibody, Anti-Bax antibody, Anti-NF-κB p65 antibody and Anti-NF-κB p65 (phospho S536) antibody were purchased from Abcam. The p38 MAPK, Phospho-p38 MAPK (Thr180/Tyr182), P44/42 MAPK (Erk1/2), and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) were purchased from Cell Signaling Technology. β-actin was severed as the internal control and anti-β-actin was purchased from Labgic Technology Co., Ltd.
9
Western Blot Analysis of DCUN1D1, NF-κB
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Cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) containing a protease inhibitor. Protein concentration was measured using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Next, the protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (40 µg/lane) and transferred to polyvinylidene fluoride) membranes (Sangon Biotech Co. Ltd.). The membranes were blocked with 5% defatted milk, followed by overnight incubation at 4˚C with primary antibodies [anti-DCUN1D1 antibody (cat. no. ab181233; 1:10,000; Abcam), anti-nuclear factor κ enhancer binding protein (NF-κB) p50 antibody (cat. no. ab32360; 1:5,000; Abcam), anti-NF-κB p65 antibody (cat. no. ab28856; 1:1,000; Abcam)] and later with HRP-conjugated secondary antibodies (goat anti-rabbit, A0208; 1:1,000; Beyotime Institute of Biotechnology) at room temperature for 2 h. The protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. ab8245; 1:1,000; Abcam).
10
RAW264.7 Cells Western Blot Analysis
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RAW 264.7 cells were seeded at 2 × 106 cells per well in 6-well plates and incubated with MSN (10 μg/mL), Len/OVA/MSN (10 mg/mL) and Len/OVA/yMSN (10 mg/mL) for 24 h. Cells incubated with PBS were used as control. Then, RAW264.7 cells were collected, centrifuged and resuspended in RIPA buffer for immunoblotting analysis. Briefly, cell lysates in sample buffer were loaded onto a proper polyacrylamide gel and subsequently transferred onto a nitrocellulose membrane. The membranes were incubated with primary antibodies at 4 °C overnight before being blocked with 5% BSA for 1 h at 28 °C. Next, the membranes were washed and incubated with a horseradish peroxidase-conjugated secondary antibody (anti-actin antibody, anti-NF-κB p65 antibody, anti-MyD88 antibody and anti-IRAK1 antibody, all antibodies purchased from Abcam) for 1 h. Before being exposed to film and developed, the membranes were incubated with an ECL substrate kit (Abcam).
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