Nf κb p65 primary antibody

MCF-7 and BT20 cells were seeded in a 96-well plate (Corning, USA) at a density of 8,000 cells/well and then treated with various concentrations (10, 32, 100 μM) of DMDD for 2 h, followed by incubation with 10 ng/ml tumor necrosis factor-alpha (TNF-α) (Sigma-Aldrich, USA) for 30 min. Untreated cells and cells treated with 10 ng/ml TNF-α alone served as controls. Cells were fixed, permeabilized, and sequentially stained with NF-κB p65 primary antibody (Cell Signaling Technology, USA), DyLight 488-conjugated secondary antibody, and Hoechst 33342 dye. The Hoechst and DyLight fluorophores detect changes in nuclear morphology (blue fluorescence) and NF-κB distribution (green fluorescence), respectively. The samples were analyzed on an Arrayscan VTI HCS Reader (Thermo Scientific, USA). The Nuclear Translocation BioApplication (Thermo Scientific, USA) was used for image acquisition and analysis. For each well, at least 400 cells were automatically acquired and analyzed. The translocation index was calculated by measuring the average intensity difference of NF-κB between the identified cytoplasmic region and nuclear region (MEAN_CircRingAvgIntenDiffCh2).

Translocation of NFκB-p65 from cytoplasm to the nucleus was detected using an immunofluorescent analysis. RAW 264.7 cells were seeded onto glass slides (Merck, Darmstadt, Germany). Following 1 h stimulation of the cells by respective samples, cells were fixed by paraformaldehyde and blocked with PBS containing 1% BSA and 0.3% Triton at room temperature for 1 h. NFκB-p65 primary antibody (Cell Signaling Technology, Denver, MA, USA) was applied overnight at 4 °C. Following removal of the primary antibody, the secondary antibody labeled with Alexa 488 (CS-4412, Cell Signaling Technology) was added for 1 h at room temperature. Images were captured using a fluorescent microscope (Oxion fluorescence, Euromex, Arnheim, The Netherlands).

Immunofluorescent analysis of NF-κB p65 was performed in RAW264.7 cells plated onto Millicell^® EZ slides (Merck KGaA, Darmstadt, Germany) and treated with 10 μmol·L⁻¹ CAPE for 30 min prior to exposure to 1% saliva. The cells were fixed in paraformaldehyde and blocked with 1% bovine serum albumin and 0.3% Triton in PBS at room temperature for 1 h. Then, the cells were incubated with a NF-κB p65 primary antibody (1:100; Cell Signaling Technology, USA). An Alexa 488 secondary antibody (1:200, Santa Cruz Biotechnology, USA) was applied for 1 h. The cells were washed, the nuclei were stained with 4′,6-diamidino-2-phenylindole (100 ng·mL⁻¹) and the cells were mounted onto glass slides. Fluorescent images were captured at ×100 in oil immersion using a Zeiss Axiovert 200M fluorescence microscope.