Ihc select detection systems

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.