Immunofluorescence on fly brains and VNCs was performed as described (67). Anti-GABA (A2052, Sigma-Aldrich) was used at a dilution of 1:500, nc82 antibody (Developmental Studies Hybridoma Bank) at a dilution of 1:75, cleaved caspase-3 antibody (Asp175, Cell Signaling Technology) at a dilution of 1:500, and anti-Twist (GTX127310, GeneTex) at a dilution of 1:500. Secondary antibodies (Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647 from Thermo Fisher Scientific) were used at dilutions of 1:500. Confocal sections were acquired using a Leica DMI 6000 SP8 confocal microscope with 40× numerical aperture (NA) 1.30 oil objective at 0.6-μm intervals and with 63× NA 1.4 oil objective at 0.34 μm. Top-view pictures were made by performing maximum projections of image stacks in ImageJ (National Institutes of Health; http://rsbweb.nih.gov/ij/), and tangential side-view images were made by using ImageJ and Leica Application Suite X (LASX) software. GABA foci and Gad1-LaminGFP–positive cells were quantified using three-dimensional object counter function in ImageJ. Leg imaging was performed at 16×/0.5 multi-immersion (IMM) objective at 2.34-μm intervals, and tarsus segment imaging was acquired at 40× oil objective at 0.6-μm intervals, of the same confocal microscope. Neuropathy of ppk+ neurons in the leg was assessed by measuring dendritic length retained in the leg using ImageJ.

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