Immunocytochemical analysis of NF-kappaB p65 (Phospho-T435) staining in K562 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with the primary antibody in 3% BSA-PBS and incubated overnight at 4 °C in a hidified chamber. Cells were washed with PBST and incubated with a DyLight 594-conjugated secondary antibody (red) in PBS at room temperature in the dark.

Immunohistochemical analysis of NF-kappaB p65 (Phospho-T435) staining in human breast cancer formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0). The section was then incubated with the antibody at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Western blotting analysis of NF-kappaB p65 (Phospho-T435) expression in Jurkat TNFa-treated (A), HepG2 TNFa-treated (B) whole cell lysates.