Neurons expressing YFP-PrPC were imaged previously17 (link). Neurons expressing PrPC-EGFP or Mito-EGFP were imaged 48 hr post-transfection using a Nikon Ti-E Perfect Focus inverted microscope equipped with a total internal reflection fluorescence (TIRF) setup, with a Andor iXon + DU897 EM Camera, and a 100×/1.49 NA oil objective. A 488 nm laser was used for detecting GFP fluorescence and it was positioned at an angle for pseudo-TIRF acquisition42 . Transfection rates were ~1%, which enabled imaging of individual axons. Movies of PrPC-EGFP vesicle transport were 15 sec long and collected at a frame rate of 10 frames/sec (10 Hz). Movies of Mito-EGFP transport were 5 min long and collected at a frame rate of 0.5 frames/sec (0.5 Hz). For all movies acquired, exposure time was set to 100 ms. Plates with cultured neurons were maintained at 37°C and 5.5% CO2 throughout the total imaging period. The pixel size was 0.126 μm for YFP-PrPC movies and 0.16 μm for PrPC-EGFP and Mito-EGFP movies, and this was adjusted accordingly in the KymoAnalyzer analyses.