Primary Neuron Culture Derivation and Staining

Primary neuron cultures were generated following procedures described previously [21 (link), 22 (link), 27 (link)]. Briefly, embryos were dechorionated using bleach, selected at approximately stage 11, sterilized with ethanol and mechanically dissociated. Cells were then chemically dispersed, washed in Schneider’s medium with 20% fetal calf serum and plated onto concanavalin A (5 μg/ml) coated glass coverslips. Coverslips were kept on custom incubation chambers, where cells were grown as hanging-drop cultures at 26 °C for 3–10 days in vitro (DIV). Primary neurons were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS; pH 6.8 or 7.2) for 30 min at room temperature and then washed three times in PBS with 0.3% Triton X-100 (PBT), followed by staining. Antibody staining and washes were performed in PBT using anti-tubulin (clone DM1A, mouse, Sigma-Aldrich, 1:1000, RRID:AB_477583) and anti–GFP (ab290, rabbit, abcam, 1:1000, RRID:AB_303395). Secondary antibodies were anti-rabbit Alexa Fluor 488 (1:1000, RRID:AB_2576217, goat) and Cy3-conjugated anti-mouse (1:200; Jackson Immuno-Research, RRID:AB_2315777, donkey). Culture slides were mounted in ProLong Gold.