AT was carried out according to previously published protocols44. Briefly, hCSs were removed from their growth medium and immediately fixed with 4% PFA, 2.5% sucrose in PBS. The tissue was embedded and sectioned into ribbons of 70 nm thick serial sections (30–50 sections/ribbon). Ribbons were stained in four consecutive sessions with the following antibodies: PSD-95 (rabbit, 1:200; Cell Signaling 34505), VGLUT1 (guinea pig, 1:5,000; Millipore: AB5905), SYN-1 (rabbit, 1:500; Cell Signaling: 52975), MAP2 (guinea pig, 1:1,000; Synaptic Systems: 188 004), GFAP (rabbit, 1:500; DAKO: Z0334), NR2B (mouse, 1:500; Neuromab: 75–101). The images were processed and registered for volume reconstruction using plugins from FIJI/ImageJ. The final volumes were rendered using AxioVision software (rel. 4.7, Zeiss).
