Neuron immunocytochemistry

Control/transfected/treated HNs were cultured on GRs up to DIV4, fixed for 15 min with 4% formaldehyde with 4% sucrose in PBS at room temperature and processed as previously reported [5]. For axon-dendrite staining, the cells were incubated with primary antibodies anti-SMI312 (mouse, 1:100; Abcam ab24574), as axonal marker, and anti-MAP2 (guinea pig, 1:500; Synaptic Systems 188004), as dendrite marker; for some samples, anti-Tau antibody (rabbit, 1:1000; Synaptic Systems 314002) was used as axonal marker. Samples were incubated in GDB buffer (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4) overnight at 4 °C and then with appropriate secondary antibodies conjugated to AlexaFluor-488 and 647 (Invitrogen, 1:150) respectively in GDB at room temperature (for 1 h). For GCs staining, HNs were incubated with mouse anti-Tubulin-α (Sigma T5168; 1:500) primary antibody together with phalloidin-AlexaFluor647 (Invitrogen A22287; 1:40). For selected experiments, to check the UBE3A expression levels in WT+UBE3A or AS+UBE3A samples, WT/AS+UBE3A HNs were fixed and processed for immunostaining with anti-UBE3A (1:500; Sigma-Aldrich E8655) and anti-MAP2 antibodies in GDB buffer and then with the appropriate secondary antibodies conjugated to AlexaFluor488 and 647, respectively (Additional file 1: Figure S2a). After final washing in PBS and H₂O_mQ, samples were mounted using Fluoroshield mounting medium with 4′,6-diamidin-2-fenilindolo (DAPI) to stain nuclei (Sigma, F6057).