Axio observer z1 invert confocal microscope

Third instar wandering larvae were dissected, fixed, antibody stained, imaged and analysed as described previously (West et al., 2015 (link)). All NMJ analysis was performed double-blind. Primary antibodies used were Cy3-Conjugated anti-HRP (Goat, 1:200, Jackson ImmunoResearch Labs Cat^# 123–165-021, RRID:AB_2338959), anti-synaptotagmin (Rabbit, 1:2000, Syt-91, RRID:AB_2713991, (West et al., 2015 (link))) anti-polyubiquitinated proteins (Mouse, 1:2000, FK2, Enzo Life Sciences Cat^# BML-PW8810–0500, RRID:AB_2051891), anti-RAB4 (Rabbit 1:100, Abcam Cat^# ab78970, RRID:AB_2042753), anti-RAB5 (Rabbit, 1:500, Abcam Cat^# ab31261, RRID:AB_882240) and anti-Spinster (Guinea Pig, 1:1000, RRID:AB_2833057, (Sweeney and Davis, 2002 (link))). Drosophila motor-neurons were labelled using GFP-tagged even-skipped (eve). Confocal microscopy was performed using a Zeiss LSM 880 on an Axio Observer.Z1 invert confocal microscope (Zeiss). Z-stacked projections of NMJs and VNCs were obtained using a Plan Neofluar 40×/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 (West et al., 2015 (link); West et al., 2018 (link)).

Quantification of synaptic bouton number at the Drosophila third instar larval neuromuscular junction (NMJ) was performed as described previously (7 (link)). 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 40×/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 (7 (link)). Corrected total cell fluorescence (CTCF) quantification was performed as described previously using ImageJ (7 (link)). Neurons were identified in the Drosophila larval VNC using anti-elav.

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)].