Triton x 10

Neurons were fixed with 4% paraformaldehyde solution on day 35. Antigen blocking and cell permeabilization were performed using 10% horse serum (Sigma), 0.2% Triton X-10 (Sigma), and 0.5% bovine serum albumin (Sigma) in PBS. Cells were incubated with primary antibodies diluted in blocking solution for 1 hour at room temperature. Fluorophore-coupled secondary antibodies were also diluted in blocking solution and were used for 1 hour at room temperature. All antibodies are listed in Supplementary Table S2. Cultures were counter-stained with 4′,6-diamidino-2-phenylindole (Sigma). Imaging was performed using Nikon Spinning Disk Confocal microscope. For imaging of presynaptic and postsynaptic markers, images were acquired with a ×100 objective in z-stacks.
For dendritic spines imaging, cells were infected on differentiation day 20 with IncuCyte NeuroLight Red Lentivirus—Synapsin Promoter (Sartorius). Cells were fixed at day 35 following 7 days of drug treatment and stained using an antibody against mKate2 expression as described above. Images were acquired using the Nikon Confocal A1R with a ×60 objective and a ×2 digital zoom in z-stacks. Dendritic and spine morphology was assessed using Neurolucida (MBF Bioscience).
Presynaptic and postsynaptic marker analysis was performed using CellProfiler and total dendritic length was measured using Fiji.