Larval Neuromuscular Junction Analysis

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)).

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West R.J., Ugbode C., Fort-Aznar L, & Sweeney S.T. (2020). Neuroprotective activity of ursodeoxycholic acid in CHMP2BIntron5 models of frontotemporal dementia. Neurobiology of Disease, 144, 105047.

Publication 2020

Cy3-Conjugated anti-HRP ab31261 LSM 880 Axio Observer.Z1 invert confocal microscope

Antibodies Antibody Confocal microscope Drosophila Goat Guinea pig Larvae Motor neurons Mouse Proteins Rabbit Synaptotagmin

Corresponding Organization : University of York

Other organizations : University of Sheffield

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Variable analysis

independent variables

Dissection of third instar wandering larvae

dependent variables

Neuromuscular junction (NMJ) analysis
Synaptic protein levels (synaptotagmin, polyubiquitinated proteins, RAB4, RAB5, Spinster)
Neuronal morphology (NMJ length)

control variables

All NMJ analysis was performed double-blind

controls

Positive control: GFP-tagged even-skipped (eve) labeling of Drosophila motor-neurons
Negative control: Not explicitly mentioned

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