Anti phospho p65 pp65

BmMs (10⁴/well) from mice with CIA treated with either PBS or 12b (1 μg/ml) were incubated for 18 h in Lab-Tek chamber slides (Nunc) before being stimulated with LPS (100 ng/ml) for 15 min then fixed with 4% formaldehyde for 15 min [15] (link). Samples were quenched with 50 mM ammonium chloride (Fisher Scientific) for 10 min, washed, permeabilised with 0.1% Triton X-100 (Sigma) in PBS for 20 min and then washed and incubated for 20 min with PBS containing 1% BSA and 10% normal goat serum (Sigma). Cells were then incubated with anti-phospho-p65 (pp65; Cell Signalling) or the relevant isotype control rabbit IgG (Santa Cruz Biotech Inc) in PBS containing 1% BSA and 10% normal goat serum overnight at 4 °C. The cells were washed, and incubated with fluorescein-conjugated anti-rabbit IgG (Vector Laboratories) at 10 μg/ml in 1% BSA, 10% normal goat serum in PBS for 1 h in the dark. Cells were washed and stained with DAPI (Invitrogen) as a counterstain. Cells were washed again, and the slides mounted in Vectashield (Vector Laboratories) for analysis by LSC. LSC data were analysed using WinCyte software (CompuCyte). Using the relocation feature of the LSC, areas with the average representative fluorescence were relocated to and digital images of the stained cells were obtained using a Hammamatsu camera and Openlab software (Improvision) [15] (link).