RAW 264.7 cells were seeded on glass coverslips in 6-well plates for 24 h. The cells were then treated with DHMDT (200–800 µg/ml) for 1 h, followed by stimulation with 500 ng/ml LPS for 30 min. Untreated RAW 264.7 cells served as the control. The cells were then rinsed twice with PBS and fixed with 3.7% paraformaldehyde in PBS for 10 min at 4°C. Following fixation, the cells were incubated with 0.4% Triton X-100 for 10 min and blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, followed by probing with rabbit anti-p65 NF-κB antibody (1:50; cat. no. sc-109; Santa Cruz Biotechnology, Inc.) overnight at 4°C. The cells were then incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (1:200; cat. no. 711-095-152; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 2 h at room temperature. After washing with PBS, the nuclei were counterstained with DAPI solution (1 mg/ml) for 15 min in the dark, and fluorescence was visualized using a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).
Rabbit anti p65 nf κb antibody
Manufactured by Merck Group
The Rabbit anti-p65 NF-κB antibody is a research-use only laboratory reagent. It is a polyclonal antibody raised in rabbits that binds to the p65 subunit of the NF-κB transcription factor complex.
Automatically generated - may contain errors
2 protocols using rabbit anti p65 nf κb antibody
1
DHMDT Suppresses LPS-Induced NF-κB Activation
2
Fluorescence Imaging of NF-κB Activation in RAW264.7 Cells
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RAW264.7 cells plated into a 4-well plate with a density of 6 × 104 cells/well were pre-treated with 150 µM of wistin for 30 min, followed by LPS (0.1 µg/mL) treatment. After 2 h, cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) in PBS for 20 min. The cells were then incubated with 0.2% Triton X-100 and 0.1% citrate (Sigma-Aldrich; Merck KGaA) in PBS for 5 min, washed with PBS, and blocked with 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 30 min, followed by probing with rabbit anti-p65 NF-κB antibody (1:500; cat. no. 8242; Cell Signaling Technology, Inc.) for 2 h. After washing with 2% bovine serum albumin, the cells were incubated with fluorescein goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 555 (4 µg/mL; cat. no. A-21428; Invitrogen Inc., Middlesex County, MA, USA) for 1 h. After washing with 2% bovine serum albumin, the nuclei were counterstained with DAPI solution (1 mg/mL) for 5 min in the dark, and fluorescence was visualized using a fluorescence microscope (Zeiss microscope, Carl Zeiss AC).
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