An avidin-biotin complex method was used for the immunofluorescence staining in the differentiated spheroid and neuronallike cell. In brief, cells were plated onto poly-L-ornithine-coated glass coverslips and fixed with 4% paraformaldehyde for 15 to 20 minutes at room temperature, and then were washed twice (10 minutes each) with 16 PBS. Cells were permeabilized with 0.1% Triton X-100/PBS for 10 minutes at room temperature, and then twice (10 minutes each) with 16 PBS. The cells were then blocked with blocking solution for 30 minutes and were incubated with primary antibodies (Oct-4, Chemicon, Temecula, CA) for 1 hour at room temperature. We then washed the cells three times (10 minutes each) with 16 PBS. Immunoreactive signals were detected with a mixture of biotinylated rabbit antimouse IgG and Fluorsave (Calbiochem, San Diego, CA). Cells were further probed with fluorescein isothiocyanate (FITC)tagged secondary antibodies. Fluorescence images were visualized with a fluorescence microscope. To quantitatively analyze the fluorescence intensity, we recorded images with an inverted fluorescence microscope equipped with a CCD camera. The percentage of signal fluorescence per photographed field was analyzed by image processing software (Image Pro-Plus, Media-Cybernetics, Inc., Silver Spring, MD).