Quantitative Analysis of Larval NMJ Structure

Third instar wandering larvae were dissected, fixed, antibody stained, imaged and analyzed as described previously [49 (link), 50 (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) and, anti-synaptotagmin (Rabbit, 1:2000, Syt-91, RRID:AB_271399). Secondary antibodies used were anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:1000, RRID:AB_2576217, goat). NMJs were imaged for quantification using an EVOS M5000 microscope. NMJ images shown were imaged using a Leica SP5 confocal microscope. NMJ bouton number and muscle surface area was quantified manually using images in ImageJ. Bouton number and length were normalized to muscle surface area. NMJ lengths were measured from stacked NMJ images using the NeuronJ plugin for ImageJ as described previously [49 (link), 50 (link)].

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Bennett C.L., Dastidar S., Arnold F.J., McKinstry S.U., Stockford C., Freibaum B.D., Sopher B.L., Wu M., Seidner G., Joiner W., Taylor J.P., West R.J, & La Spada A.R. (2023). Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity. Acta Neuropathologica Communications, 11, 164.

Publication 2023

Cy3-Conjugated anti-HRP SP5 confocal microscope

Alexa fluor 488 Anti igg Antibodies Antibody Confocal microscope Goat Larvae Microscope Muscle Rabbit Synaptotagmin

Corresponding Organization : University of Sheffield

Other organizations : Duke University, St. Jude Children's Research Hospital, University of Washington Medical Center, University of California, San Diego

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Variable analysis

independent variables

Third instar wandering larvae

dependent variables

NMJ bouton number
Muscle surface area
NMJ length

control variables

Experimental conditions (dissection, fixation, antibody staining, imaging, analysis) as described previously in references [49] and [50]
All NMJ analysis was performed double-blind

controls

Positive control: Cy3-Conjugated anti-HRP (Goat, 1:200, Jackson ImmunoResearch Labs Cat# 123-165-021, RRID:AB_2338959)
Negative control: Not explicitly mentioned

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