Ab23345

The SVZ tissue was processed for both P14 and 12-month-old mice as described above (104 (link)). To identify the different subpopulations of NPCs, the following primary antibodies were used: Sox2 (1:400 Novus Biologicals, catalog AF2018), Tbr2 (1:200 Abcam, catalog AB23345), and Ki67 (1:500 Thermo Fisher Scientific, catalog 14-5698-82), followed by secondary antibodies (all Invitrogen at 1:1,000: donkey α-rat Alexa Fluor 488, catalog A-21208; 1:1000, donkey α-rabbit Alexa Fluor 555, catalog A-32794; 1:1000, donkey α-goat Alexa Fluor 647, catalog A-32849). All tissue was counterstained with DAPI. 3D 20 μm Z-stack images were acquired (×20 magnification) on a Zeiss Axio Imager.M2 with an ApoTome.2 system. Z-projections were produced using ZEN 2.6 (blue edition). For further details, see Supplemental Methods.

Western blotting was performed according to standard procedures using peroxidase-conjugated secondary antibodies and SuperSignal^® West Pico chemiluminescent substrate (Thermo). Primary antibodies were EOMES (Abcam #ab23345, 1:1000), GAPDH (Thermo #AM4300, 1:10,000), and NKX2.5 (R&D #AF2444, 1:100). All uncropped immunoblots can be found in Supplementary Fig. 5.

Deparaffinized sections of P18 mice were incubated for 20 minutes in citrate buffer (10mM, pH 6.0) at 98°C in a steam bath for antigen retrieval, washed with PBS and permeabilized by PBS with 0.1% Tween 20, blocked with 10% BSA and incubated with primary antibody at 4°C overnight. The following primary antibodies were used: mouse anti-Ap2α (Santa-Cruz, 12726X, 1:3000), mouse anti-Calbindin-D-28K (Sigma-Aldrich, clone CB-955, C9848, 1:2500), mouse anti-Lim1/2 (DSHB, clone 4F2-c, 1:300), rabbit cone arrestin (Millipore, AB15282, 1:1000), rabbit anti-Pax6 AF1 (1:3000), rabbit Sox9 (Millipore, 1:500), rabbit anti-Tbr2 (Abcam, ab23345, 1:500), sheep anti-Chx10 (ThermoFisher Scientific, PA1-12565, 1:500). Next day, sections were washed with PBS, incubated with fluorescent secondary antibody Alexa Fluor 488 or 594 (Life Technologies, 1:500) for 1 hour at room temperature, washed with PBS, counterstained with DAPI (1 μg/ml) and mounted in mowiol (4–88; Sigma).