Zen 2 lite imaging software profile tool

Fluorescent images were obtained on a Carl Zeiss widefield epi-fluorescence microscope via AxioVision camera and software. Low-magnification images for stage analyses were taken at 20× while high-magnification images for neuron measurements were taken at 40× or 100×. Image analysis was completed through ImageJ software. We used the NeuronJ software plugin to trace and measure axons and minor neurites. Axon branches were only counted if they were a minimum of 5 μm. Neurite lengths were measured from the base of the process at the soma to the tip of the Tuj1 stain. Stage 3 neurons’ minor neurites were measured in thickness at 0 μm, 5 μm, and 10 μm from the edge of the soma. Stage 3 neurons’ axons were measured in thickness at 0 μm, 5 μm, 10 μm, and 25 μm from the edge of the soma. For Fig. 7D, images of tubulin in axons were captured using a Deltavision widefield microscope and then deconvolved. The intensity of tubulin staining was measured across the width of the axon at 5 μm, 10 μm, and 25 μm using Zeiss Zen 2 lite imaging software profile tool.

Images of Shootin1 immunostaining were captured using the Zeiss AxioImagerZ1 Microscope with a 100× oil objective, and a constant 600 milliseconds exposure time. Tau and DCX images were captured using the 40× objective on the Zeiss Observer Z1 microscope with Axiocam 506 mono camera. Exposure time was kept constant (DCX 300 ms and Tau 200 ms). Zeiss Zen 2 lite imaging software profile tool was used to measure intensity. Excel was used to compare individual cells line scans as well as create averages across genotypes for comparison.