Larvae were reared and dissected, as previously described (54 ), and at least 20 NMJs (muscles 6–7, hemisegment A2) per genotype per stage were scored (55 (link)). For immunohistochemistry, anti-discs large (DLG, DSHB, 4F3), anti-HRP (Jackson Immuno, 323-005-021), anti-bruchpilot (DSHB, nc82) and anti-HA (Santa Cruz, sc-805) were all used at 1/100, with AlexaFluor 488 goat anti-rabbit and AlexaFluor 633 goat anti-mouse (Invitrogen) secondary antibodies at 1/1000. Z-stacks were taken using a Leica SP5 laser confocal microscope and analyses performed using ImageJ and Photoshop (Adobe). For BRP analysis in the axon, optical sections of 0.2 µm were taken using a laser-scanning confocal microscope (Leica TCS SP5 II confocal microscope). Bouton size analysis was performed by averaging two perpendicular diameter measurements of individual Ib boutons. All observable Ib boutons, which were identified by intense DLG staining, were measured per NMJ. All digital analyses and fluorescence measurements were performed using ImageJ. For BRP and HA staining at the synapse, average fluorescence intensity was analysed over the whole synapse (marked by HRP staining), again using optical sections of 0.2 µm. For HA staining, this was normalized to the background fluorescence intensity in the muscle.
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