Immunocytochemistry

The cortical cultures were washed with phosphate buffer solution (PBS) for 5 min and fixed with paraformaldehyde (4% in PBS and 2% sucrose) for 15 min, followed by thrice washing with PBS for 5 min each. Background immunoreactivity was blocked and cells permeabilized with 10% fetal bovine serum (FBS) in Triton X-100 0.1% for 1 h. This step was followed by overnight incubation of cultures with chicken anti-microtubule associated protein 2 (map2) (1:10000; Abcam, ab5392), mouse anti–PSD-95 (1:500; Abcam, ab192757), anti–glial fibrillary acidic protein (1:500; GFAP, rabbit polyclonal, 1:5000; Dako, catalogue # Z033429), or guinea pig anti–synapsin I/II (1:1000; Synaptic Systems, 106004). Dendritic spines were stained using Alexa Fluor 405 Phalloidin, which is known to label F-actin enriched in spines (Thermo Fisher, catalogue #A30104) (Fig. 1G-I). After 3 washes, fixed samples were incubated with the secondary donkey-anti-chicken, donkey-anti-mouse, and donkey-anti-guinea pig antibodies conjugated to Alexa Fluor 488, 594, and 647 (1:500; Jackson ImmunoResearch, 703–545-155, 715–605-150, and 706–585-148, respectively) for 1 h followed by rinsing with PBS for 5 min and mounting with FluoroShield (Sigma-Aldrich, F6182), for viewing and confocal imaging.