Coverslips were fixed with 4% PFA or ice-cold methanol (95% methanol, 5% HAC) after 49 (direct contact) or 56 (indirect contact) days of differentiation. The immunocytochemistry staining process in short, coverslips were blocked for 1.5 hour at RT in blocking buffer (5% NGS, 0.1% BSA, 0.3% TritonX-100 in PBS 1X) then incubated with primary antibody in blocking buffer for 1 hour at RT followed by overnight incubation at 4°C (primary antibodies are listed in Table A in S1 File). After washes (3X for 5 minutes each in PBS 1X) the coverslips were incubated with secondary antibody in blocking buffer at RT for 1 hour, and mounted with Fluoromount G (Southern Biotech) on glass slides (VWR; Super frost plus). Images were acquired using a Carl Zeiss 510 Meta confocal microscope with 40x (1.2 Numerical Aperture (NA)) and 63x (1.4 NA) oil objectives. Synaptophysin1, VGAT and VGLUT1 puncta analyses were performed with 63x oil objective images using SynD [26]. A minimum of 10 fields of views (FOVs) was obtained per coverslip. And, synapses were analysed using SynD with threshold parameters of 0.5,
