Human iNs were fixed for 15 minutes in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS at room temperature. Cells were premeabilized in 0.2% Triton X-100 in PBS for 10 minutes and then incubated in blocking buffer (1× PBS, 20% Goat serum) for 1 hour 30 minutes. Cells were then incubated overnight with primary antibodies. The primary antibodies used were: anti-MAP2 (Sigma, M3696, 1:750; Sigma, M1406 1:1,000 and 1:500), anti-vGlut2 (NeuroMab clone N29/29, 1:1,000), anti-tyrosine hydroxylase (TH, Millipore AB152, 1:500), anti-GAD6 (Sigma, G5038, 1:1,000), anti-β-III-tubulin (Tuj1, Covance, 1:500), anti-synapsin (rabbit anti-synapsin, E028, a gif from the Südhof lab, 1:3,000) and Hoechst (1×). Excitatory (glutamatergic) neurons were stained for MAP2, vGlut2 and β-III-Tubulin. Dopaminergic neurons were stained for MAP2 and TH. Inhibitory (GABAergic) neurons were stained for GAD6. Confocal images were collected using a Zeiss LSM700 laser scanning microscope (Carl Zeiss, Dublin, CA). Three-way circuit images were acquired by taking Z-stacks and processing the stacks as maximum intensity projections.
