Immunolabeling of Embryonic Brain Tissues

Immunofluorescence staining was performed as described previously^{19 (link)}. For immunohistochemistry, GD 18 embryo brains were dissected and fixed with 4% paraformaldehyde (PFA) for 18 h. After fixation, brains were washed with phosphate buffered solution overnight and incubated with a 30% sucrose solution for 18 h for cryoprotection. The following antibodies were used: rabbit anti-Sox2 (Abcam, Cambridge, MA; 1:1000), mouse anti-Tuj1 (Millipore, Burlington, MA; 1:1000), and rabbit anti-Calbindin D (Swant, Switzerland; 1:2000). For immunocytochemistry, cells were fixed with 4% PFA, then stained with rabbit anti-Sox2 (Abcam, Cambridge, MA; 1:1000) and mouse anti-Tuj1 (Millipore, Burlington, MA; 1:1000). For F-actin staining, Alexa Fluor 488 conjugated phalloidin (Invitrogen, Carlsbad, CA; 1:1000) was used. Appropriate fluorophore-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) were used with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Abcam, Cambridge, MA) for nuclear staining. All images were acquired using an LSM-800 confocal microscope with ZEN software (Zeiss, Oberkochen, Germany).