Gold anti fade mounting solution

Brains were dissected in PBS and fixed in PBS with 4% paraformaldehyde at room temperature for 20 min. They were washed three times, 20 min each in PBS containing 0.5% Triton-X100 (PBT), followed by 30 min incubation in PBT containing 5% normal goat serum. Anti-GFP (1:1000; Invitrogen, A11122), anti-TH (1:200; Millipore, AB152) and anti-nc82^{39 (link)}(1:50; DSHB) antibodies were added to the solution and brains were incubated overnight at 4°C. Brains were then washed in PBT three times, 20 min each at room temperature, followed by incubation in PBT containing Alexa 488 conjugated goat anti-rabbit (1:100; Invitrogen, A11034) and Cy3 conjugated goat anti-mouse (1:200; Jackson ImmunoResearch, 115-165-003) overnight at 4°C. Brains were then washed in PBT three times, 20 min each at room temperature, before being mounted on slides with Gold anti-Fade mounting solution (Invitrogen). Imaging was performed using a Leica TCS SP5X confocal microscope. The resolution of the image stack was 1024 × 1024 with 1μm step size. Images were processed using Fiji. For each genotype, at least two brains were dissected to confirm that they had the same pattern of expression.