Brains of different groups of 30-day-old flies were dissected in cold PBS and fixed in 4% paraformaldehyde in PBS for 1 h. The samples were washed and permeabilized overnight in 0.3% Triton X-100 in PBS (Wash buffer). Fixed brains were stained with mouse anti-TH antibody (mouse monoclonal, Immunostar)/anti-α-syn antibody (Syn1- Rabbit polyclonal, BD Biosciences)/anti-α-syn filament antibody (rabbit monoclonal, cat number ab209538; Abcam) at a 1:200 dilution for 48 h. After incubation, the brains were washed three times and incubated with the corresponding secondary antibodies overnight (anti-mouse Alexa 488/anti-rabbit Alexa 594) at 1:1000 dilution. After thorough washing, the stained brains were mounted using Fluoromount mounting medium (Sigma), and images were acquired using a confocal microscope (Nikon EZC1). DA neuron clusters were analyzed as previously described [38 (link)]. Optical sections of the brains were acquired at 40-μm intervals using a 40× objective for whole-brain imaging. Confocal stacks were merged into a single plane using Nikon EZC1 software (Nikon, Tokyo, Japan). The number of TH-positive neurons within the PPL1 and PPL2 DA neuronal clusters was counted by visual inspection of the individual confocal Z-series of images. An average of eight brains were analyzed in both hemispheres, for each genotype, and the results are expressed as the mean ± S.E.M.
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