AT was used to collect high-resolution images of synapses within neural spheroids using previously published protocols11,54. Briefly, fused hSS-hCS were fixed in 4% paraformaldehyde in phosphate buffered saline. To preserve GFP fluorescence, the tissue was dehydrated with up to 70% alcohol only, with processing through 50% ethanol, 70% ethanol, 1:3 70% ethanol:LR White Resin (LRW, medium grade, SPI supplies), and LRW overnight before embedding in LRW. The embedded tissue was sectioned into ribbons of 70 nm thick sections (~30 sections/ribbon) and each ribbon was immunostained in 2–3 rounds of staining with the antibodies eluted after each round. The following primary antibodies were used for immunostaining: anti–GFP (chicken, 1:200; Genetex: 13970, 1:200), anti–SYN1 (rabbit, Cell Signalling: 5297S, 1:500), anti–PSD95 (rabbit, Cell Signalling: 3450S), anti–VGUT1 (guinea pig, 1:5000; Millipore: AB5905), anti–Gephyrin (mouse, 1:100; BD Biosciences: 612632), anti–VGAT (guinea pig, 1:200; Synaptic Systems 131004), anti–VGAT (mouse, 1:200; Synaptic Systems: 131011), anti–GFAP (chicken, 1:300; Aves), anti–MAP2 (guinea pig, 1:1000; Synaptic Systems: 188004). Sections were visualized on a Zeiss Axio Imager.Z1 upright fluorescence microscope using AxioVision software (rel 4.7, Zeiss). Images were processed and registered using FIJI/ImageJ with standard and custom plugins (code.google.com/p/smithlabsoftware). FIJI/ImageJ was used for volume reconstruction.
