Anti ap1

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.

Both primary and BV-2 microglial cells, control and treated, were washed with 1× phosphate-buffered saline (PBS) thrice and were fixed with acetone to methanol followed by permeabilization with 0.3 % Triton X-100 in PBS (PBST). Cells were then incubated with anti-α-tubulin (1:500) or anti-NF-kB (1:300) or anti-AP1 (1:300) or anti-HSP-70 (1:300) (from Sigma-Aldrich) diluted in 2 % BSA, for 24 h at 4 °C in humid chamber. After two to three washings with 0.1 % PBST, the cells were incubated with the secondary antibody anti-mouse IgG 488 and anti-rabbit IgG 488 (prepared in 2 % BSA (1:500)) for 2 h at room temperature. Cells were incubated with (DAPI, 1:5000 in 1× PBS) for 10 min for nuclear staining and then mounted with anti-fading reagent (Fluoromount, Sigma). For CellRox and Mitotracker staining, after treatment period, the dye was added in culture according to the manufacturer’s instructions; cells were then fixed and mounted. Images were captured using Nikon AIR Confocal Laser Microscope and analyzed using NIS elements AR analysis software version 4.11.00. Experiment was performed in triplicate.