Neurons were fixed 3–5 days after plating in the microchambers. A 2 min prelysis was done using 0.5% saponine in PBS, after which cells were fixed for 20 min using 4% paraformaldehyde and 4% sucrose in PBS. Once fixed, microchambers were rinsed three times with PBS and blocked for 1 h at room temperature with 3% BSA+0.3% Triton X-100. Primary antibodies were incubated overnight at 4 °C in blocking buffer and secondary antibodies were incubated for 1 h at room temperature.
Super-resolution images were taken in a Leica SR GSD (Leica Microsystems, Mannheim, Germany) GSD microscope, equipped with a HCX PL APO × 100 objective (Leica Microsystems) with a 1.47 NA for TIRF illumination. Images were acquired on a sensitive electron-multiplying CCD iXon3 camera (ANDOR, Belfast, UK). The lasers used were 405, 488, 532 and 642 nm diodes with approximate powers of 30 mW, 300 mW, 1 W and 500 mW, respectively (Coherent, Santa Clara, CA, USA).
High-resolution images were acquired using a Zeiss LSM 710 inverted confocal microscope using Airyscan detector, to improve signal to noise and resolution, and a × 63 objective (1.4 NA, Zeiss).
Super-resolution images were taken in a Leica SR GSD (Leica Microsystems, Mannheim, Germany) GSD microscope, equipped with a HCX PL APO × 100 objective (Leica Microsystems) with a 1.47 NA for TIRF illumination. Images were acquired on a sensitive electron-multiplying CCD iXon3 camera (ANDOR, Belfast, UK). The lasers used were 405, 488, 532 and 642 nm diodes with approximate powers of 30 mW, 300 mW, 1 W and 500 mW, respectively (Coherent, Santa Clara, CA, USA).
High-resolution images were acquired using a Zeiss LSM 710 inverted confocal microscope using Airyscan detector, to improve signal to noise and resolution, and a × 63 objective (1.4 NA, Zeiss).