Fluorescent confocal image of SY5Y cells stained Phospho-Nanog-S71 antibody. SY5Y cells were fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min), then incubated Phospho-Nanog-S71 primary antibody (1:500, 2 h at room temperature). For secondary antibody, Alexa Fluor 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Cytoplasmic actin was counterstained with Alexa Fluor 555 (red) conjugated Phalloidin (5.25 mu M, 25 min). Phospho-Nanog immunoreactivity is localized very specifically to the nuclei of the SY5Y cells.