Immunohistochemistry of P1 Mouse Brain Sections

Newborn mice were sacrificed at P1 and brain removed, fixed overnight in PBS 4% paraformaldehyde, cryoprotected in 20% sucrose, and rapidly frozen by immersion in isopentane on dry ice. For each brain, a complete set of coronal sections was cut through the SN and VTA at 30 μm. Sections were mounted on slides, blocked, and permeabilized with PBS, 5% BSA, 0.1% Triton for 30 min at RT. They were incubated with an anti-TH rabbit polyclonal antibody diluted in PBS, 1% BSA, overnight at 4°C, followed by a biotinylated goat anti-rabbit antibody (Jackson ImmunoResearch) diluted in PBS, 1% BSA for 30 min at RT, and by the ABComplex and development with DAB. All reagents are detailed in Supplementary Table S4.