Goat anti sox2

Transverse sections were processed as described (11 (link)). Idu/BrdU antigens were exposed by 40min DNAseI treatment at 37 °C, except mouse E8.5 where 2N HCl was used.
Flat mount preparation: the neural tube was cut at the roofplate, fixed in 4% PFA and subsequently methanol. Antibody incubations and washes were 24h each. The left/right neural tube halves were split, then mounted with grease spacers between slide and coverslip.
Sections were imaged with 40×/1.25NAOil objective, flat mounts with 20×/0.7NADry on a Leica TCS-SP5-MP. Single optical sections were taken, except for GBS-GFP and pSmad analysis, where a maximum projection of 3 z-slices 1 μm apart were used. For flat mounts, the entire apicobasal depth of the progenitor layer was imaged with z-slices 1.5 μm apart.
Antibodies used were: goat anti-Sox2 (R&D, 1:100), rat anti-pH3 (Novus Biologicals, 1:2000), rabbit anti-Olig2 (Millipore, 1:1000), rabbit anti-Arx (50 (link)) (from J. Chelly, 1:1000), mouse anti-Pax3(c) (DSHB, 1:100), rabbit anti-Islet1 (51 (link)) (from T. Jessell, 1:3000), sheep anti-GFP (Biogenesis, 1:1000), mouse anti-Nkx2.2 (DSHB, 1:25), rabbit anti-mAzami Green (MBL, 1:100), rabbit anti-cleaved Caspase3 (Cell Signaling, 1:500); rabbit anti-pSmad1/5/8 (from Ed Laufer), mouse anti-BrdU/IdU (1:80, BD clone B44), rat anti-BrdU (1:80, Abcam, clone BU1/75).

Immunocytochemical staining was performed following the protocol described before (Zhang et al., 2001 (link)). Antibodies used included rabbit anti-Oct4 (1:200, Abcam, Cambridge, MA, http://www.abcam.com), goat anti-Sox2 (1:1,000, R&D, Minneapolis, MN, http://www.rndsystems.com/), mouse anti-Pax6 (1:5,000, DSHB, Iowa City, IA, http://dshb.biology.uiowa.edu/), rabbit anti Olig2 (1:500, Chemicon & Millipore, Billerica, MA, http://www.millipore.com), mouse anti Hb9 (1:50, DSHB), goat anti ChAT (1:300, Chemicon & Millipore), Rabbit GFAP (1:5000, DAKO, Carpinteria, CA, http://www.dako.com), rat anti MAP2 (1:1000, Chemicon & Millipore), mouse anti hGFAP (1:500, Stem Cells Inc, Newark, CA, http://www.stemcellsinc.com/).
Images were collected with a Nikon TE600 fluorescence microscope (Nikon Instruments, Melville, NY) or a Nikon C1 laser-scanning confocal microscope (Nikon, Tokyo, Japan). The populations of MAP2/GFP, hGFAP /GFP positive cells in the spinal cord were counted in fields chosen by an automated stage movement operated by Stereo Investigator software (MicroBrightField Inc.) or using Z-section images analyzed using image J software. GFP positive neurons or GFP positive astrocytes were counted every six sections as described (Ma et al., 2012). For cell quantification in vitro, 150-200 neurons were counted from 3 repeated experiments. Data are presented as mean ± SEM.