Ultraview scanner

All live-cell imaging for rab11 vesicle movement studies was performed using an Olympus IX70 microscope equipped with a CCD camera (ORCA-ER, Hammamatsu, Japan) and a PerkinElmer UltraVIEW scanner for spinning disk confocal microscopy. MetaMorph software was used to operate the system.
Rab11 vesicle movements were imaged at the proximal axon and dendrite; the first 100 μm segment was considered as proximal. Images were taken one frame/second for 3 min. Kymographs were obtained from the most well focused 30 μm strip of the axon or dendrite. Vesicles moving only in one direction for more than 2 μm were defined as either anterograde (towards the distal axon) or retrograde (towards the cell body). Vesicles moving in both directions over 2 μm was termed as bidirectional. The average speed for every anterograde or retrograde movement during the imaging period was quantified, and their average was reported as the cell’s anterograde or retrograde vesicle velocity. All analysis was performed with FIJI.

Live-cell imaging was performed using spinning disk confocal microscopy, using an Olympus IX70 microscope with a Hamamatsu EM-CCD Image-EM camera and a PerkinElmer Ultra-VIEW scanner. Videos were taken using Meta-Morph software (Version 7.6.1.0). Rab11 and integrin vesicle trafficking along the axon was imaged at the proximal (up to 100 μm) and distal part (beyond 600 μm) of axons of neurons transfected with Protrudin (as described before in^{18 (link),29 (link)}). In short, one image per second was obtained for 3 min. Kymographs were obtained by measuring an individual axon segment. Anterograde, retrograde, bidirectional and static modes of transport were measured. The percentage of co-localization between integrin or rab11 and Protrudin was calculated as the number of vesicles containing either was divided by the total number of vesicles. All analysis was performed using Meta-Morph software (Version 7.6.1.0).

Laser-scanning confocal microscopy was performed using a Leica DMI4000B microscope, with laser scanning and detection achieved by a Leica TCS SPE confocal system controlled with Leica LAS AF software. Fluorescence and wide-field microscopy was performed using a Leica DM6000B with a Leica DFC350 FX CCD camera and a Leica AF7000 with a Hamamatsu EM CCD C9100 camera and Leica LAS AF software. Leica AF7000 was also used for imaging of axon and growth cone regeneration after axotomy. Live confocal imaging was performed with an Olympus IX70 microscope using a Hamamatsu ORCA-ER CCD camera and a PerkinElmer UltraVIEW scanner for spinning disc confocal microscopy, controlled with MetaMorph software.