Live-Cell Imaging of Axonal Transport

For live imaging, cells were maintained in Hibernate E medium without phenol red (Brain-Bits). Images were acquired using a microscope (model Ti-E; Nikon) equipped with a spinning-disk confocal-head (model CSUW1, Yokogawa), and images were captured with an sCMOS camera (Zyla, Andor). KIF3AB and KIF3AC images were acquired with an Andor Dragonfly built on a Ti2 (Nikon) with a CFI Apo 60× 1.49 objective (Nikon) and captured with an sCMOS camera (Zyla 4.2+, Andor). The entire imaging stage and objectives were maintained at 37°C in a warmed enclosure (full lexan incubation ensemble; OkoLab). A Plan-Apo 100× 1.49 NA objective (Nikon) was used with 2 × 2 binning to acquire image streams. During image acquisition, z-axis movement was controlled by the Perfect Focus system on the Ti-E microscope (Nikon). Recordings were acquired at two frames per second. For further details, see Kaech et al.⁸² Axons were identified with anti-neurofascin antibody (NeuroMab, Cat #: 75–027) conjugated to CF405 (Mix-n-Stain CF405S Antibody Labeling Kit; Biotum, Cat#: 92231) in the imaging medium. Cells expressing constructs with HaloTag were treated with 50 nM Janelia Farm 549 dye^{18 (link)} for 10 minutes and washed with conditioned medium for 10 minutes prior to imaging.