Ab5603

The cultured aNSCs were fixed with 4 % parafromaldehyde for 30 min at room temperature, followed by washing with cold PBS for 15 min. The cells were blocked with 3 % goat serum and 0.1 % TritonX-100 in PBS for 1 h at room temperature, followed by the incubation with primary antibodies at 4 °C overnight. The second day, the cells were incubated with secondary antibodies after washed with PBS for 30 min. The images were taken with a Zeiss confocal microscope. Primary antibodies: Rabbit SOX2 (Millipore, Ab5603), Mouse Nestin (BD, #556309). Secondary antibodies: goat anti Rabbit 488 (Invitrogen, A11008), goat anti Mouse 568 (Invitrogen, A11004).

2D NPC cultures and cells on microcarriers were fixed with 4 % PFA in PBS, permeabilized with 0.1 % Triton-X100 (Sigma) for 15 min, blocked with 10 % donkey serum (Dianova, Ref. 017-000-121) for 30 min. Subsequently, cells were incubated with either rabbit anti-Sox2 (Millipore, AB5603, 1:400) and mouse anti-nestin (BD Biosciences, 611658, 1:250), rabbit anti-PCNA (abcam, ab18197, 1:1000) and mouse anti-nestin (BD Biosciences, 611658, 1:250) or rabbit anti-GFAP (Dako, Z0334, 1:250) and mouse anti-Map2 (Sigma, M1406 in. 1:1000) in blocking solution at 4 °C over-night. After washing with 1X DPBS three times, secondary antibody incubation was performed with donkey anti-rabbit Alexa Fluor568 IgG (H + L) (Life technologies, A10042, 1:500) and donkey anti-mouse Alexa Fluor488 IgG (H + L) (Dianova, 715-545-151, 1:500) in blocking solution for 3 h at room temperature in the dark. Unbound secondary antibody was washed away with 1X DPBS. Thereafter, cells were counterstained with Hoechst 33342 nuclear dye (Molecular Probes, Invitrogen, 1:1000 in DPBS) for 10 min and again washed with 1X DPBS.

NSPCs were plated at 5 × 10⁴ cells/ml on poly‐D‐lysine treated coverslips. Cells were fixed with 4% paraformaldehyde for 10 min, blocked for 1 h with 5% goat serum/0.1% BSA, followed by incubation for 2 h at room temperature with primary antibody (SOX2 [1:200, EMD Millipore AB5603], NESTIN [1:200, BD Pharmingen #556309]). After washing five times with PBS/0.05% Tween‐20, coverslips were incubated with the appropriate secondary antibody for 1 h at room temperature (Molecular Probes Alexa Fluor, goat anti‐rabbit 488, goat anti‐mouse 546). Cells were imaged with a Zeiss Axiovert 200 M Fluorescence microscope.