Rabbit monoclonal nfκb antibody

Cells were washed with PBS twice and fixed for 10min in PBS solution containing 4% paraformaldehyde and 4% sucrose, and permeabilized with 0.25% triton-100× for another 10min at room temperature. After washing 3 times with PBS, cells were blocked with 10% horse serum for 1h at room temperature. Next, cells were incubated overnight at 4°C with primary antibodies prepared in 10% blocking solution containing 0.1% tween-20. Primary antibodies used including mouse monoclonal P-gp antibody C219 (Covance; MA) at 1:50 dilution, and rabbit monoclonal NFκB antibody (Abcam, MA) at 1:100 dilution. Subsequently cells were washed three times with PBS and incubated for 1h at room temperature with secondary antibodies either Alexa Fluor 488 donkey anti-mouse IgG (Thermo Fisher Scientific Cat# R37114, RRID:AB_2556542) or Alexa Fluor 546 donkey anti-rabbit IgG (Thermo Fisher Scientific Cat# A10040, RRID:AB_2534016) at 1:200 dilutions followed by washing cells three times with PBS. Transwell membranes were collected and mounted with Prolong Gold DAPI antifade solution (Life technologies, CA) and images were captured using Olympus Fluoview 1000 Confocal laser scanning microscope (Olympus, PA) at a total magnification of 400×. Total signal of P-gp was quantified using NIH ImageJ version 1.44 software as described previously (Noursadeghi et al., 2008).