Raw data collection was performed as previously described7 (link). Briefly, young-adult hermaphrodite C.elegans expressing the synaptic vesicle precursor marker RAB-3 in the DA9 motor neuron (wyIs251[Pmig-13::GFP::RAB-3]) were paralyzed in 0.3 mM Levamisole in M9. Once paralyzed, worms were carefully transferred to M9 solution on a 10% agarose pad for imaging. This results in an effective Levamisole concentration which is significantly lower than the concentration that was suggested to affect axonal transport42 (link). Worms were maintained on the pad for no more than 20 min, although we confirmed that viability was maintained even after 4 h.
Fluorescence imaging was performed using a Nikon 60 × CFI plan Apo VC, NA 1.4 objective on a Nikon Ti-E microscope equipped with Yokogawa CSU-X1 scan-head and a Hamamatsu C9100-50 EM-CCD camera at a frame rate of 110 ms/frame, and 240 nm per pixel.
Post-analysis fluorescence movies were corrected and analyzed in imageJ. Animal movement was corrected using FIJI plugin StackReg. Kymographs were generated with KymoBuider. Intensity was averaged ± 5 pixels transverse to the kymograph line.
Fluorescence imaging was performed using a Nikon 60 × CFI plan Apo VC, NA 1.4 objective on a Nikon Ti-E microscope equipped with Yokogawa CSU-X1 scan-head and a Hamamatsu C9100-50 EM-CCD camera at a frame rate of 110 ms/frame, and 240 nm per pixel.
Post-analysis fluorescence movies were corrected and analyzed in imageJ. Animal movement was corrected using FIJI plugin StackReg. Kymographs were generated with KymoBuider. Intensity was averaged ± 5 pixels transverse to the kymograph line.
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