Anti sox1

Human NPCs were characterized by immunocytochemistry. Briefly, cells grown on glass coverslips were fixed with 4% paraformaldehyde and incubated in the blocking buffer (5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100) for 15 min. Cells were then incubated in primary antibodies diluted in the 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, anti-SOX2 (1:200, R&D Systems), anti-Nestin (1:500, R&D Systems), anti-SOX1 (1:250, Millipore). 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.

For in-situ immuno-staining, cultured hESCs and iPS cells were fixed with 4% para-formaldehyde in PBS for 20 min and washed with PBS for 15 min. For embryoid body (EB) formation assays, EBs of 8 days were transferred onto gelatin-coated plates for additional 2-day attachment (after mild pipetting to break the EBs into smaller pieces). The fixed samples were incubated with the following primary antibodies for 2 hours at room temperature: anti-TRA-1-60 (Millipore), anti-SOX1 (Millipore), anti-OCT-4 (Millipore), anti-SSEA-4 (Millipore), anti-αSmooth Muscle Actin (Sigma), anti-Tubulin beta III isoform (Millipore), anti-AFP (Sigma). After wash with PBS, Alexa-488 or Texas Red conjugated goat anti-rabbit or anti-mouse secondary antibodies (1∶500, Invitrogen) were used for 1-hour incubation to visualize the cells together with DAPI nuclear staining.