An immunostaining experiment is detailed in Figure 4 . Procedures match those previously published (Brusich et al., 2015 (link), 2018 (link); Spring et al., 2016 (link); Yeates et al., 2017 (link); James et al., 2019 (link)). Briefly, third instar larvae were filleted and fixed for 5 min with Bouins fixative (Ricca Chemical, Arlington, TX, USA). After washes, fixed fillets were incubated in primary antibodies overnight at 4C, mouse anti-Brp (nc82, 1:250, University of Iowa Developmental Studies Hybridoma Bank; Wagh et al., 2006 (link)) and rabbit anti-Dlg (1:5,000; Budnik et al., 1996 (link)). After washes, fillets were incubated in fluorophore-conjugated secondary antibodies overnight at 4C (Jackson ImmunoResearch Labs, West Grove, PA, USA), goat anti-mouse-488 (DyLight, 1:1,000) and goat anti-rabbit-549 (DyLight, 1:2,000). After washes, fillets were mounted and Dlg boutons were counted blinded by hand on an epifluorescence microscope and double checked for Brp signal in apposition. Note: relative bouton numbers between NMJs 6/7 on segment A2 and A3 are consistent with earlier studies, though some raw numbers appear slightly lower, which may be due either to hand counting (rather than automated) or due to Dlg signal bouton counting (rather than HRP signal counting).
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