Nf kb p65 primary antibody

Immunofluorescence analysis was performed on gingival fibroblasts treated with IL1β and TNFα at a concentration of 5 ng/mL for 20 minutes before being exposed to 100 µM UDCA for 1 hour. Cells were fixed in paraformaldehyde and blocked in 1% BSA and 0.3% Triton in PBS for 1 hour at room temperature. Cells were subsequently incubated with nuclear factor (NF)kB p65 primary antibody¶

Cell Signaling Technology Europe, Frankfurt am Main, Germany.

and Alexa 488 secondary antibody#

Santa Cruz Biotechnology, Dallas, TX.

was applied for 1 hour. Cells were washed and mounted onto glass slides. Images were captured at 100× in oil immersion using a fluorescence microscope.

Immunofluorescent analysis of nuclear factor-kappa B (NF-kB) p65 was performed in HGF and HSC2 cells plated onto Millicell® EZ slides (Merck KgaA, Darmstadt, Germany) at 1 × 10⁴ cells/cm². Cells were submitted to overnight serum starvation and then stimulated with IL1β + TNFα or the cell lysates for 1 h. Cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO), blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO), and permeabilised with 0.3% TritonX-100 (Sigma-Aldrich, St. Louis, MO). Cells were subsequently incubated with NF-kB p65 primary antibody (1:400, Cell Signaling Technology, Cambridge, UK) at 4 °C overnight. Detection was carried out by incubation of Alexa 488 secondary antibody (1:800, Cell Signaling Technology, Cambridge, UK) for 1 h at room temperature. Fluoromount-G mounting media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) containing DAPI was used to mount the glass slides. Images were captured under a fluorescent microscope with a dual excitation filter block DAPI-FITC (Echo Revolve Fluorescence microscope, San Diego, CA) to observe the nuclei translocation.