After exposure to TGF-β1 and silibinin, the cells were fixed with 4% paraformaldehyde for 20 mins, permeabilized with 1% Triton X for 20 mins. After blocking with 5% skim milk, the cells were incubated with antibody against NF-κB (ab131546, 1:200, Abcam, Cambridge, MA, USA) at 4°C overnight. On the second day, the cells were incubated with goat-anti-rabbit IgG H&L secondary antibody, ab150077, 1:1,000, Abcam, Cambridge, MA, USA for 50 mins at 37°C, then incubated with DAPI for 10 mins at 37°C in the dark. Immunofluorescence was visualized under fluorescence microscope (Olympus, Tokyo, Japan).
Ab131546
Manufactured by Abcam
Sourced in United States
Ab131546 is a laboratory equipment product. It is designed for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (1 mentions)
Goat anti rabbit igg h l secondary antibody, by Abcam (1 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Ab23875, by Abcam (1 mentions)
Ab3759, by Abcam (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab54481, by Abcam (1 mentions)
Ab137550, by Abcam (1 mentions)
Ab13243, by Abcam (1 mentions)
Ab133625, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab124957, by Abcam (1 mentions)
Ab7217, by Abcam (1 mentions)
Ab59195, by Abcam (1 mentions)
Ab97931, by Abcam (1 mentions)
3 protocols using ab131546
1
NF-κB Immunofluorescence Assay
2
Cardiac Protein Expression Analysis
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The cardiac tissues were harvested, washed three times in sterile saline and then homogenized in RIPA buffer (Beyotime Institute of Biotechnology, Suzhou, People’s Republic of China) containing a protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Following centrifugation at 16,000 × g at 4°C for 30 mins, the supernatant was collected. Protein was quantified using a BCA Protein Assay kit (Beyotime Institute of Biotechnology), then separated by 10% SDS-PAGE. Following transfer to a PVDF membrane (Beyotime Institute of Biotechnology), the primary antibodies were then incubated at 4°C overnight. Subsequently, they were incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit (ZB-2301) secondary antibodies (ZSGB-BIO) for 1 hr at room temperature and visualized under a chemiluminescence system (Bio-Rad, USA). The bands were quantified using MultiGauge version 3.2 software. Relative expression levels of proteins were calculated by integrated grey values of the bands normalized with reference protein. The primary antibodies: AMPK (1:800; ab3759, Abcam, Boston, MA, USA), p-AMPK (Thr 172) (1:500; ab23875, Abcam), Nrf2 (1:800; ab137550, Abcam), HO-1 (1:1000; ab13243, Abcam), PGC-1α (1:800; ab54481, Abcam), NF-κB (1:800; ab131546, Abcam).
3
Placental Protein Expression Analysis
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Frozen placenta tissues and collected cells were taken out from liquid nitrogen and lysed with RIPA lysis and extraction buffer (89901, Thermo Fisher Scientific, USA). The manufacturer's instruction was strictly followed. The protein samples were collected from the supernatants after centrifugation. Protein samples were then loaded into SDS-PAGE gels to run for electrophoresis, followed by PVDF membrane transfer and skim milk blocking processes. Postblocking membranes were incubated at room temperature with primary antibodies against PPARα (ab24509, Abcam, USA), CD36 (ab133625, Abcam, USA), acyl-CoA oxidase (ACO) (ab248375, Abcam, USA), uncoupling protein 2 (UCP2) (ab97931, Abcam, USA), NF-κB (ab131546, Abcam, USA), IKKβ (ab124957, Abcam, USA), p-IKKβ (ab59195, Abcam, USA), IκBα (ab7217, Abcam, USA), and p-IκBα (ab133462, Abcam, USA). The primary antibodies against GAPDH (ab181602, Abcam, USA) and histone H3 (ab1791, Abcam, USA) were separately used as loading controls in total cell proteins and nucleus proteins. An HRP-conjugated goat anti-rabbit secondary antibody (ab6721, Abcam, USA) was applied to the membranes after the removal of primary antibodies, followed by an incubation of 1 hour at room temperature. SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Thermo Fisher Scientific, USA) was used during the grey analysis by using an imaging system.
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