ICC/IF image of ab125943 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 (ab124431, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.
All lanes : Anti-14-3-3 zeta antibody (ab124431) at 1 µg/mlLane 1 : Forskolin-Treated A431 Whole Cell LysateLane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell LysateLane 3 : Forskolin-Treated NIH 3T3 Whole Cell Lysate Lane 4 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 6 : HEK293 (Human embryonic kidney cell line) Whole Cell LysateLane 7 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell LysateLane 8 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.