Immunohistochemical Analysis of Dopaminergic Neurons

To determine whether neurons in the SS56699 split GAL4 line expressed TH, immunohistochemical processes was performed as described in Aso et al., 2014b (link). Briefly, 10 GFP-expressing brains were fixed (2% paraformaldehyde in Schneider’s medium), permeabilized in PBT (0.5% Triton X-100 in PBS), and blocked (5% normal goat serum for 90 min). Subsequently, brains were incubated in primary antibodies (diluted in 5% serum in PBT at 4°C for 2–4 days). Chicken anti-GFP (Abcam ab13970; 1:1000); anti-TH mouse monoclonal (Sigma MAB318; 1:200); anti-TH rabbit polyclonal (Sigma AB152; 1:200). After several washes (PBT for 30 min), brains were then incubated in secondary antibodies (diluted in 5% serum in PBT at 4°C for 2–4 days). Alexa 488 anti-chicken IgY (Invitrogen A11039; 1:400); Atto 647N anti-mouse IgG (Rockland 610-156-121; 1:400); Alexa 568 anti-rabbit IgG (Invitrogen A11036; 1:400). Finally, brains were washed thoroughly (PBT four times for 30 min or longer) and mounted on glass slides for confocal imaging.