Cells were fixed with 4% paraformaldehyde (Sigma) for 15 min at room temperature. Samples were permeabilized and blocked with 0.25% Triton X-100 (Sigma) and 10% donkey serum in PBS for 20 min as previously described16. Samples were then incubated with primary antibodies (Supplementary Table 1b) at 4°C overnight, followed by incubation with secondary antibodies for 1 h at room temperature. Antibodies were prepared in PBS containing 0.25% Triton X-100 and 10% donkey serum. Images were taken by Zeiss LSM 710 confocal microscope, or Zeiss Axiovert 200M microscope, and analysed with ImageJ (NIH). Images were acquired with identical settings for parallel cultures. For analysis of synaptic bouton density, total SV2+ puncta in a given image were counted by ImageJ Analyze Particles, and the total dendritic length were measured by ImageJ plugin NeurphologyJ35. The numbers of SYN1/PSD95 pairs were manually counted. The synaptic density was determined by D (D = total SV2+ puncta or SYN1/PSD95 pair per 100 μm total dendritic length).
