All lanes : Anti-14-3-3 zeta (phospho S58) antibody (ab51109) at 1/500 dilutionLane 1 : NIH/3T3 cell extract (treated with UV for 30 mins) with phosphopeptide immunogen (phospho S58)Lane 2 : NIH/3T3 cell extract (treated with UV for 30 mins)

ICC/IF image of ab51109 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51109, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.