Guinea pig anti dcx

Chicken anti-GFAP (Millipore, Catalog # AB5541, diluted 1:500)^{13 (link)}, Rabbit anti-NeuN (Cell Signaling Technology, Catalog # 12943, diluted 1:400)^{13 (link)}, Rat anti-BrdU (Biorad, Catalog # OBT0030G, diluted 1:200)^{30 (link)}, Guinea pig anti-DCX (Millipore, Catalog # AB2253, diluted 1:1000)^{30 (link)}, Rabbit anti c-Fos (Synaptic Systems, Catalog # 226 003, diluted 1:10,000)^{13 (link)}

Immunohistochemistry was conducted as described (16 (link)). The embryos were immersion-fixed in 4% paraformaldehyde (PFA). The postnatal and adult mice were transcardially perfused and fixed by 4% PFA. 30% sucrose was used for cryoprotection, and 12~14 μm frozen sections were made by a cryostat. Air dried slides were permeabilized by 0.3% Tween-20 in PBS, blocked by 10% lamb serum, incubated with primary antibodies at 4°C overnight. After washing in PBS, the corresponding secondary antibodies (Alexa Fluor 488 or 594 conjugated anti-rabbit, anti-mouse, anti-guinea pig, or anti-goat, 1:500, Invitrogen) were incubated for 2 hours at room temperature. Cell nuclei were stained by DAPI. The primary antibodies and dilutions are as following: rabbit anti-Olig2 (1:200, Millipore), mouse anti-Sox2 (1:50, Cell Signaling Technology), rabbit anti-Fabp7 (BLBP) (1:1000, Millipore), mouse anti-BrdU (1:50, Developmental Studies Hybridoma Bank), guinea pig anti-Dcx (1:1000, Millipore), and goat anti-OMP (1:1000, Wako). For acute BrdU labeling, pregnant mice were intraperitoneally injected with 10 mg/kg BrdU. Embryos were sampled after 1-hour BrdU incorporation. Frozen sections were treated by 2N HCl to denature DNA at 37°C for 20 min, and by 0.1M borate sodium buffer (pH 8.5) to neutralize the sections for subsequent immunohistochemistry with BrdU antibodies.

Immunostaining for guinea pig anti-DCX (1:500, Millipore), rabbit anti-DCX (1:1000, Cell Signaling Technology), and rat anti-Ki67 (1:200, Invitrogen) was carried out as described in Supplementary Information. All immunohistochemical measurements were done by blinded observers.

Mice were intracardially perfused with 4% paraformaldehyde (PFA; in 0.1 M PBS; pH 7.4) 90 minutes after the last testing session, and brains were harvested and post-fixed overnight in 4% PFA at 4°C. Brains were sectioned on a vibratome at 50 μm in the sagittal plane, blocked in 0.1 M PBS containing 10% serum and 1% Triton-X-100 (Sigma) for one hour at room temperature. For staining of BrdU, sections were washed in 2 N HCl for 10 minutes followed by three 5 minute washes in 0.1 M Sodium Borate (pH 8.5), followed by blocking. Sections were incubated with primary antibodies in 0.1 M PBS containing 1% serum and 0.3% Triton-X-100 overnight at 4°C, followed by washing in PBS and incubation with appropriate secondary antibodies for 1–2 hours at room temperature for visualization the next day. Antibodies and stains used: goat anti-cFos (Santa Cruz; 1:1000), chicken anti-GFP (Abcam; 1:2000), rabbit anti-RFP (Abcam; 1:1000), rabbit anti-HA (Cell Signaling; 1:500), mouse anti-NeuN (Millipore; 1:1000), mouse anti-BrdU (Becton Dickinson, 13:1000), guinea pig anti-Dcx (Millipore, 1:1000), and DAPI (nuclear stain; Sigma; 1:2000). Labeled sections were imaged on Fluoview 1000 (Olympus) or C1 (Nikon) confocal microscopes and double labeling was confirmed by z-stack analyses in the IMARIS software.