Ab22035

For the detection of pluripotency and differentiation markers, cells grown in 24-well plates as described above were fixed with 4% paraformaldehyde for 20 min. After that, cells were permeabilized and blocked with 10% donkey serum and 0.1% Triton X-100 in PBS. Subsequently, cells were stained with primary antibodies overnight at 4°C and finally incubated with Fluorochrome-labeled secondary antibodies ALEXA FLUOR (Invitrogen). The primary antibodies used for detecting pluripotency markers were anti-OCT4 (SC-5279, Santa Cruz Biotech), anti-SOX2 (AF2018, R&D), and anti-NANOG (AF1997, R&D). The primary antibodies used for detecting endoderm markers were anti-SOX17 (AF1924, R&D), anti-CXCR4 (MAB173-100, R&D), and anti-GATA4 (SC-25310, Santa Cruz Biotech). The primary antibodies used for detecting mesoderm markers were anti-Brachyury (AF2085, R&D), anti-EOMES (Ab23345, Abcam), and anti-MIXL1 (SC-98664, Santa Cruz Biotech). The primary antibodies used for detecting neuroectoderm markers were anti-NESTIN (AB22035, Abcam), and anti-SOX1 (AF3369, R&D) anti-SOX2 (AF2018, R&D). The secondary antibodies used were donkey anti-goat AF488 (Invitrogen), donkey anti-mouse AF488 (Invitrogen), and donkey anti-rabbit AF488 (Invitrogen). Images were captured and quantified using a Cellomics array scan, and images were processed using Adobe Photoshop CS5.

Lentivirus injected brain sections were post-fixed with 4% PFA for 10 min. Then both lentivirus injected and hNPC injected brain sections were washed with PBS three times and incubated 30 min with blocking solution (PBS/0,1% Triton X-100 containing 10% normal goat serum [Sigma-Aldrich]) and then incubated overnight at 4 °C in blocking solution with primary antibodies: mouse anti-Flag (Sigma, F3165–2 MG, 1:1000); rabbit anti-red fluorescence protein (RFP) (Invitrogen, R10367, 1:1000); rabbit anti-PAX6 (Abcam, ab5790, 1:50); mouse anti-Nestin (Abcam, ab22035, 1:100), rabbit anti-Ki67 (Abcam, ab15580, 1:1000) and mouse anti-human nuclear antigen (Abcam, ab191181, 1:1000). Sections were washed with PBS and incubated for 1 h at room temperature with the secondary antibodies: goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher, A31560, 1:500); goat anti-rabbit IgG TRITC (Abcam, ab6718, 1:1000) and goat anti-mouse IgG Alexa Fluor 647 (Invitrogen, A32728, 1:1000) diluted in blocking solution. The sections were washed with PBS and mounted with Vectashield Antifade Mounting Medium (Vector Labs, H-1000). Immunofluorescence was visualized and imaged with a Keyence BZ-X810 microscope.