Quantification of Neuromuscular Junction Morphology in Drosophila

For analysis of neuromuscular junction (NMJ) phenotypes in Drosophila, wandering third instar larvae were dissected as previously described (Smith and Taylor, 2011 (link)). Dissected larvae were fixed in 3.7% formaldehyde in PBS for 20 min, washed in PBT (PBS with 0.4% Triton X-100) and blocked with 10% normal goat serum for 1 hr at room temperature. The larvae were then incubated overnight at 4°C with mouse anti-Brp antibody (1:50; Developmental Studies Hybridoma Bank, nc82; RRID:AB_2314865) to label the active zones and Cy3 goat anti-HRP (1:200; Jackson ImmunoResearch, 123-165-021; RRID:AB_2338959) to label the NMJ membrane and axons. Next, samples were washed in PBT and incubated with Alexa Fluor 488 goat anti-mouse (1:200; Molecular Probes, A11001; RRID:AB_2534069) in PBT + 10% normal goat serum at room temperature for 2 hr. Multiple confocal stacks were acquired using a point scanning confocal microscope (Leica TCS SPE) and then processed using the DeadEasy Synapse ImageJ plugin (Sutcliffe et al., 2013 (link)), which provides total voxel occupancy by Brp signal at each nerve terminal. Brp signal was quantified at muscle 6/7 NMJs from abdominal segment A3.