Live cell imaging and photoactivation experiments were performed on an inverted Nikon Eclipse Ti‐E confocal microscope equipped with a perfect focus system (Nikon), a CSU‐X1‐A1 Spinning Disc unit (Yokogawa) and a Photometrics Evolve 512 EMCCD camera (Roper Scientific) while using a Plan Apo VC 100× N.A.1.40 oil objective. Neurons were kept at 37°C and 5% CO2 in a stage incubator (Tokai Hit) during imaging. Movies used to determine MT growth velocity were acquired at a frame rate of 1 frame per second, and acquisitions lasted 3 min per movie.
For photoactivation experiments, we made use of an ILas FRAP unit (Roper Scientific) and a Vortran Stradus 405 nm (100 mW) laser. The photoactivation procedure of paGFP‐α‐Tubulin was previously described45 . We co‐transfected neurons with GW2‐mRFP to label transfected neurons and identified axons based on morphology. We placed the soma just outside the field of view, which spanned 34 μm in total, and photoactivated axon regions of approximately 6–7 μm wide in the centre of the image. To prevent excessive photobleaching, frames were acquired at intervals varying between 6 and 15 min.
For photoactivation experiments, we made use of an ILas FRAP unit (Roper Scientific) and a Vortran Stradus 405 nm (100 mW) laser. The photoactivation procedure of paGFP‐α‐Tubulin was previously described