Immunolabeling and Imaging of Microtubules

After fixation, the samples were washed 5 times with PBST [1× PBS, 0.05% (vol/vol) Tween-20] for 10 min. The samples were blocked in 5% BSA in PBST for 1 h at RT. Primary antibody (1:500 dilution, mouse Anti-α-Tubulin Cat # T9026, Sigma-Aldrich) incubation was performed in a blocking solution (1% BSA in PBST) for 24–36 h at 4 °C. The samples were washed with PBST for 5 × 5 min, after which samples were incubated with secondary antibodies (1:250 dilution; Goat anti-Mouse IgG Alexa Fluor 594 Cat # A-11032, ThermoFisher) diluted in blocking solution for overnight at 4 °C. Then, the samples were washed with PBST for 5 × 10 min. Imaging was performed on Leica TCS SP8 DLS and Leica DMi8 confocal microscopes. Sample preparation for scanning electron microscopy was performed as in (Kraus et al. 2016 (link)); SEM imaging was performed using the JEOL IT 300 scanning electron microscope.