Tra 1 81

The cells were washed with PBS, fixed in 4% formaldehyde at RT for 15 min, and permeabilized with 0.1% Triton X-100 in PBS at RT for 20 min. After blocking with 4% normal donkey serum (Abcam, Cambridge, UK) at RT for 1 h, the samples were incubated at 4 °C overnight with primary antibodies against OCT4 (R&D Systems, 1:300), NANOG (Abcam, 1:300), SSEA-4 (R&D Systems, 1:300), Tra-1-81 (Millipore, Billerica, MA, 1:300), Tra-1-60 (Millipore, 1:300), TSP-1 (Santa Cruz Biotechnology, 1:300), KDR (Cell Signaling Technology, 1:400), VECAD (Abcam, 1:400), or Gb3 (GeneTex, Irvine, CA, 1:100) diluted with blocking solution. After rinsing several times with PBST (0.1% Tween-20 in PBS), the samples were incubated with Alexa-488- or Alexa-594-conjugated secondary antibodies (Invitrogen) at RT for 1 h. Then, the cells were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and visualized on a fluorescence microscope (Olympus, Tokyo, Japan) or a Zeiss LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany).

A2B5+ cells were characterized by immunocytochemistry (Liu et al., 2004 (link)). Briefly, cells grown on glass coverslips were fixed with 2% paraformaldehyde and incubated in blocking buffer (5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100) for 30 min. Cells were then incubated in primary antibodies diluted in blocking buffer at 4 °C overnight. Appropriate secondary antibodies were used for single and double labeling. All secondary antibodies were tested for cross-reactivity and nonspecific immunoreactivity. The following primary antibodies were used, OCT4 (1:500, Abcam), SOX2 (1:200, R&D Systems), SSEA4 (1:10, Developmental Studies Hybridoma Bank, DSHB), TRA1-81 (1:100, Millipore), A2B5 (1:20, ATCC), β3 tubulin (1:1000, Sigma), HB9 (1:10, DSHB), GABA (1:200, Sigma), NG2 (1:200, Millipore), PDGFRα (1:200, BD), CD44 (1:200, Millipore), S100B (1:200, Sigma), GFAP (1:4000, DAKO), huNA (1:200, Millipore), NFM (1:200, Sigma), MAP2 (1:200, Sigma). Bis-benzamide (DAPI, 1:1000; Sigma) was used to visualize the nuclei. Images were captured using a Zeiss Axiovision microscope with z-stack split view function.