Immunostaining of Drosophila Larvae

Immunostaining of Drosophila larvae was carried out as described previously [31 (link)]. Briefly, third-instar (L3) larvae were dissected in chilled in Calcium-free HL3 solution [79 (link)], fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 30 min, permeabilised in 0.1% Triton X-100 in PBS and incubated with the appropriate antibodies. LDs were stained with 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene 493/503 (BODIPY™, Thermo Fisher Scientific, Cat. No. D3922). Fixed and stained preparations were mounted in Vectashield (Vector Laboratories) and viewed using an Olympus FluoView FV100 confocal microscope. Images were acquired using a 60 × /1.5 NA objective and using FV10-ASX version 04.01 software. Unless otherwise stated, axonal analyses were conducted by imaging motor axon bundles passing through segment A7 [Additional file 2: Supplementary Fig. 1, online resource]. Similarly, analyses of neuromuscular junctions (NMJs) were conducted by imaging muscles 6 and 7 at segment A7.