Live staining was performed by adding primary antibodies (GCTM-2 and TG343, a kind gift from Dr. M. Pera, and TRA1-60, a kind gift from Dr. P. Andrews; SSEA-4 from Developmental Studies Hybridoma Bank [Iowa City, IA, http://www.uiowa.edu/∼dshbwww/list)] to hESCs for 20 minutes at 37°C. The primary antibodies were used at the following dilutions: GCTM-2, 1:2; TG343, 1:2; TRA1-60, 1:10; SSEA-4, 1:5. The samples were gently washed three times with ES medium before being incubated with the secondary antibodies (antimouse immunoglobulin G and anti-mouse immunoglobulin M, both 1:100 dilution [Sigma]) conjugated to fluorescein isothiocyanate (FITC) at 37°C for 20 minutes. The samples were again washed three times with ES medium and subjected to fluorescence microscopy. For OCT-4 immunostaining, hESCs were fixed in 3.7% formaldehyde (BDH, Coventry, U.K.) for 20 minutes at room temperature, followed by incubation in 3% hydrogen peroxide for 10 minutes. The hESCs were permeabilized with 0.2% Triton ×100 (Sigma) diluted in 4% sheep serum (Sigma) for 30 minutes at 37°C. The hES colonies were incubated with the primary antibodies (OCT-4 from Santa Cruz Biotechnologies, Heidelberg, Germany, http://www.scbt.com) to a final concentration of 10 μg/ml for 30 minutes at room temperature. The hES colonies were washed twice with PBS for 5 minutes and then incubated with the secondary antibody (biotinylated rat antimouse immunoglobulin [DAKO, Cambridgeshire, U.K.] used at 1:100 dilution) for 30 minutes at room temperature. After that, hESCs were washed again with PBS, incubated with avidinbiotin complex/horseradish peroxidase (ABC/HRP) solution for 25 minutes at room temperature, and washed again with PBS. The detection was carried out by incubation with diaminobenzidine (DAB) solution (Sigma) at room temperature for 1 minute. Final washes were done with distilled water. The primary antibody was omitted for the negative control. The alkaline phosphatase (AP) staining was carried out using the Alkaline Phosphatase Detection Kit following manufacturer's instructions (Chemicon International, Temecula, CA, http://www.chemicon.com). Briefly, cells were fixed in 90% methanol and 10% formamide for 2 minutes and then washed with rinse buffer (20 mM Tris-HCl at pH 7.4, 0.05% Tween-20) once. Staining solution (Naphthol/Fast Red Violet) was added to the wells, and plates were incubated in the dark for 15 minutes. The bright field images were obtained using a Zeiss microscope and AxioVision software (Carl Zeiss, Jena, Germany).