Prominin 1

Tissues were fixed in 4% PBS-buffered paraformaldehyde overnight and then transferred to 30% sucrose (w/v) for another 24 hours. Tissue was then embedded in Optimal Cutting Temperature (OCT) compound and sectioned at 8 microns on a Leica cryostat. Sections were blocked (PBS, 5% goat serum, 0.1% TritonX-100), incubated with primary antibodies for 2 hrs at room temperature in humidity chamber, washed with PBS and incubated with secondary antibodies for 45 min room temperature. Primary antibodies used: GFP (1∶250; Aves Labs Cat# GFP-1020 RRID:AB_10000240), Gabra6 (1∶100; EMD Millipore Cat# AB5610 RRID:AB_91935), NeuN Clone A60 (1∶100; Millipore Cat# MAB377 RRID:AB_2298772), BLBP (1∶250; EMD Millipore Cat# ABN14 RRID:AB_10000325), Calb1 D28K (1∶100; Sigma-Aldrich Cat# C9848 RRID:AB_476894), Olig2 (1∶150; EMD Millipore Cat# AB9610 RRID:AB_570666), Ki67 (1∶100; Abcam Cat# ab15580 RRID:AB_443209), Rspo1 (1∶1000; Sigma-Aldrich Cat# SAB3500046 RRID:AB_10602510), Pax6 (1∶250; Covance Cat# PRB-278P), Prominin-1 (1∶100; eBioscience Cat# 14-1331-90), Nestin (1∶300; BD Biosciences Cat#556309). Species-specific Alexa-Fluor-conjugated secondary antibodies were used for detection (1∶250; Invitrogen). tdTomato expression was visualized directly.