Third instar wandering larvae were dissected, fixed, antibody stained, imaged and analysed as described previously [13 (link)]. All NMJ analysis was performed double-blind. Primary antibodies detailed in Table 1 . Confocal microscopy was performed using a Zeiss LSM 880 on an Axio Observer.Z1 invert confocal microscope (Zeiss). Z-stacked projections of NMJ's and VNCs were obtained using a Plan Neofluar 40x/0.75 NA oil objective. NMJ lengths were measured from stacked NMJ images using the NeuronJ plugin for ImageJ (National Institutes of Health) as described previously [13 (link),54 (link)].
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