Anti reelin

For Reelin and NeuN immunohistochemistry, frontal, parietal and retrosplenial brain sections of each fetus were selected (Fig. 1B). Endogenous peroxidase of free-floating sections was inactivated twice for 15 min at room temperature (RT) with H₂O₂ at 6% in PBS. After 3 washes with PBS, sections were immersed in blocking solution (IHC Select Detection Systems, Millipore) for 2 hours at RT, and then incubated with the monoclonal anti-Reelin (1:1000, Millipore) and anti-NeuN (1:100, Millipore) as primary antibody, over 2 nights at 4°C. After the incubation and the corresponding washes with PBS, brain sections were incubated for 1 hour at RT with the biotynilated anti-mouse secondary antibody (IHC Select Detection Systems, Millipore), then and after subsequent washes with PBS, sections were incubated with Streptavidin-HPR (IHC Select Detection Systems, Millipore) for 1 hour at RT. After washing, the staining was revealed using diaminobenzidine (1:400, IHC Select Detection Systems, Millipore). Finally, sections were cleaned in distilled water and mounted.

Immunohistochemistry was performed essentially as in [10 (link)]. Brains were dissected, fixed, embedded and sectioned as for ISH, except for fixation in PFA4% that was often 3–4 h at 4°C. Sections were washed in PBS 5 min, unmasked in citrate buffer (Na Citrate 0.01 M, Citric acid 0.01 M pH6) by boiling in a microwave 3 min and then washed in PBS 10 min at RT. Sections were blocked with blocking solution (FBS 10%, Triton 0.3%, PBS1X) for 1 h at RT, then incubated O/N in blocking solution with primary antibodies: anti-mSOX2 (R&D Systems MA2018, 1 : 50), anti-P73 (Neomarkers, 1:150), anti-Reelin (Millipore MAB5364, 1:500), anti-Tuj1 (Covance, 1:400), anti-GFP (Invitrogen A10262, 1 : 500, used to detect EYFP expressing cells), anti-GFAP (Dako, 1:500). Slides were then washed in PBS two times, 10 min each, and incubated in blocking solution containing the secondary fluorescent antibody (1 : 1000, Alexa Fluor Invitrogen) for 1 h 30 min at RT. Slides were then washed in PBS twice, 10 min each, and then mounted with Fluoromount (F4680, Sigma) with 4′,6-diamidino-2-phenylindole (DAPI) and imaged with a confocal microscope (Nikon A1R) and with a Zeiss Axioplan 2 fluorescent microscope for anti-GFAP immunostainings.