Immunolabeling of Neuronal Cytoskeleton

Neurons were seeded on poly-L-lysine-coated #1.5 coverslips (633029; Carolina Biological Supply). At indicated timepoints, coverslips were briefly washed in Dulbecco’s PBS with calcium and magnesium (PBL02; Caisson Labs), and fixed for 15 min with 4% paraformaldehyde (diluted in PBS from 16%, Electron Microscopy Services, 15710) and 4% sucrose in PBS at 37°C. For immunolabeling of fascin, coverslips were washed and fixed for 10 min with ice-cold methanol at −20°C. Subsequent steps were performed at room temperature. Autofluorescence was quenched by incubation with 50 mM NH₄Cl for 10 min at room temperature. Coverslips were blocked and permeabilized using a buffer of 10% goat serum and 0.1% Triton X-100 in PBS and incubating for 30 min. Antibodies were diluted in a buffer of 3% goat serum and 0.1% Triton X-100 in PBS. Coverslips were incubated with primary antibodies for 1 h, washed three times in PBS with 0.1% Tween-20, and incubated with secondary antibodies and Alexa Fluor 488 phalloidin (A12379; Thermo Fisher Scientific, 1:40) for 30 min. Coverslips were mounted using Prolong Gold Antifade (P36930; Thermo Fisher Scientific), and allowed to cure for at least 24 h prior to imaging. Antibodies used were as follows: mouse monoclonal anti-Arp2/3 complex (#MABT95; Millipore, RRID:AB_11205567; 1:250), mouse monoclonal anti-fascin (#54545; Cell Signaling Technology, RRID:AB_2799464; 1:50), rabbit monoclonal anti-β-III-tubulin (#5568; Cell Signaling, Clone D71G9, RRID:AB_10694505; 1:100), rabbit monoclonal anti-MAP2 (#8707; Cell Signaling, Clone D5G1, RRID:AB_2722660; 1:500), goat anti-rabbit Alexa Fluor 568 (#A-11011; Molecular Probes, RRID:AB_143157; 1:1,000), goat anti-rabbit Alexa Fluor 647 (#A-21245; Thermo Fisher Scientific, RRID:AB_2535813; 1:1,000).