Ab51109

The day after seeding, cells were washed three times with PBS and fixed with 4% formaldehyde (Serva) in PBS for 10 min at room temperature followed by a wash with PBS and permeabilization in PBS containing 0.1% Triton X-100 for 10 min at room temperature. Cells were then washed with PBS and blocked in blocking buffer (5% goat serum (Biosera) in PBS) for 1 h at room temperature. After blocking, cells were incubated with a primary antibody (Anti-14-3-3ζ (phospho-S58) antibody (ab51109, Abcam) and 14-3-3ζ Antibody (MA5-37641, Invitrogen)) diluted in blocking buffer at 1:200 for 1 h at room temperature. Then the cells were washed 4 × 5 min with PBS supplemented with 0.05% Tween 20 (PBS-T) and incubated with secondary anti-mouse antibody conjugated with Alexa Fluor 488 (Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488), ab150113) and anti-rabbit antibody conjugated with Alexa Fluor 594 (Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594), ab150080) diluted in blocking buffer at 1:250 for 1 h at room temperature. Finally, the cells were washed 4 × 5 min with PBS-T and mounted on microscope slides using ProLong Omega Glass (Invitrogen) with DAPI staining for nuclear DNA.