Cortical neurons were fixed with a 4% paraformaldehyde (Sigma-Aldrich)/4% sucrose (Wako) solution, permeabilized with 0.25% Triton X-100 (Nacalai), and blocked in a 0.5% solution of fish skin gelatin (Sigma-Aldrich). Axons, somatodendritic regions, and presynaptic sites were stained with SMI-312 [837904, BioLegend; mouse immunoglobulin G1(IgG1)/IgM], MAP2 (ab5392, Abcam; chicken IgG), and Syn1 (106 001, Synaptic Systems; mouse IgG1) antibodies, respectively. The secondary antibodies were Alexa Fluor 488–labeled goat anti-mouse IgG1 (A21121, Molecular Probes) and Alexa Fluor 568–labeled goat anti-chicken IgG (A11041, Molecular Probes). Fluorescence images were obtained using either an epifluorescence microscope (IX83, Olympus) or a confocal microscope (SP8, Leica).

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