Anti ncam

Control and treated cells were given a washing with chilled 1X PBS followed by fixation with acetone and methanol (1:1) and permeabilization with 0.3% Triton- X100 in 1X PBS. Cells were then blocked with 2% BSA and incubated with primary mouse monoclonal antibody anti-α-Tubulin (1:500), anti-NF-κB (1:500), anti-MAP-2 (1:250), anti-NF200 (1:500), anti-GAP 43 (1:250), anti-HSP70 (1:500), anti-Mortalin (1:500), anti-Bcl-xL (1:200), anti-Cyclin D1(1:250), anti-NCAM (1:250), rabbit monoclonal anti-AP-1(1:250) (all from Sigma-Aldrich) and mouse monoclonal anti-PCNA (1:250), mouse polyclonal anti-PSA-NCAM (1:250) (from Millipore, MA, USA) for 24 h in humid chamber at 4 °C. No permeabilization was carried out for PSA-NCAM immunostaining. After primary antibody incubation, three washings were given with 0.1% PBST and incubated with secondary antibody (goat anti-mouse/ rabbit IgG/ IgM Alexa Fluor 488/543) for 2 h at RT. Cells were stained with nuclear staining dye DAPI (Sigma-Aldrich) for 15 min, washed with 0.1% PBST and mounted with antifading agent Fluoromount (Sigma-Aldrich). Images were captured with Nikon AIR Confocal Laser Scanning Microscope and analyzed with NIS elements analysis software version 4.11.00 (Nikon Co., Tokyo, Japan). Each experiment was carried out in triplicate.