Immunostaining of Drosophila Brains

Immunostaining was performed as described^{50 (link)}. Fly heads were removed and fixed in PBS with 4% paraformaldehyde and 0.008% Triton X-100 for 45–50 min at 4°C. Fixed heads were washed in PBS with 0.5% Triton X-100 and dissected in PBS. The brains were blocked in 10% goat serum (Jackson Immunoresearch, West Grove, PA) and subsequently incubated with primary antibodies at 4°C overnight or longer. For VGLUT and GFP co-staining, a rabbit anti-DVGlut (1:10000) and a mouse anti-GFP antibody (Invitrogen; 1:1000) antibody were used as primary antibodies. For GRASP staining, a mouse anti-GFP monoclonal antibody (Invitrogen; 1:1000) and a rabbit anti-GFP antibody (Roche; 1:200) were used. After washing with 0.5% PBST three times, the brains were incubated with Alexa Fluor 633 conjugated anti-rabbit and Alexa Fluor 488 conjugated anti-mouse (Molecular Probes, Carlsbad, CA) at 1:500 dilution. The brains were washed three more times before being mounted in Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA) and viewed sequentially in 1.1 μm sections on a Leica SP5 confocal microscope. To compare the fluorescence signals from different conditions, the laser intensity and other settings were set at the same level during each experiment. Fluorescence signals were quantified by ImageJ as described.