Brain dissection and immunohistochemistry

Brain dissection and immunohistochemistry were performed according to the Janelia Flylight protocols available online at https://www.janelia.org/project-team/flylight/protocols. Adult fly brains were dissected in cold Schneider’s Insect Medium (S2) and fixed for 24 h in 1.2% paraformaldehyde (PFA) in S2 at 4°C. After four 10 min washes in 0.5% Triton-X100 in PBS (PBT) at 4°C, samples were blocked and permeabilized in 5% normal goat serum (NGS) in 0.5% PBT for 1.5 h at room temperature (RT). Samples were then incubated in a primary antibody solution of mouse anti-dDAT (1:1000; gift from Dr. Eric Gouaux) in 5% NGS for 3.5 h at RT and 2 overnights at 4°C. After a rinse and three 30 min washes in PBT at RT, brains were incubated in a secondary antibody solution of Alexa Fluor 488 goat anti-mouse IgG (1:500; Invitrogen) in 5% NGS in PBT for 3.5 h at RT and 2 overnights at 4°C. Secondary antibodies were then removed, samples were rinsed and then washed for three 30 min washes at RT in PBT. For pre-embedding fixation, PBT was replaced with 1.75 mL of 4% PFA in PBS for 4 h at RT. Fix was then removed and samples were rinsed and then washed for four 15 min washes at RT.

To mount the samples for confocal imaging, glass microscope slides (Fisher Scientific, Premium Superfrost, 75 × 25 mm) were prepared with two glass coverslips (Fisher Scientific, No. 1, 22 × 22 mm) placed 1 cm apart and secured with glycerol to prevent flattening of the brain sample when mounted. Whole-mount brains were then mounted to a coverslip (Thermofisher Scientific, No. 1.5, 20 × 20 mm) with ProLong Gold antifade mountant (Invitrogen) and the slide was placed on top. Slides were allowed to cure for 48 h in the dark prior to imaging.