Immunohistochemistry of Drosophila Brains

Immunohistochemistry was performed as described previously30 (link). Briefly, 3–7-day old brains were dissected in ice-cold PBS, fixed in 4% paraformaldehyde, and blocked in PBST (PBS and 0.3% Triton X-100) with 5% normal donkey serum (Jackson Laboratory) before staining. Brains were then incubated with 1:1,000 rabbit anti-GFP (Invitrogen) and 1:50 mouse anti-nc82 (Developmental Studies Hybridoma Bank) antibodies overnight at 4 °C and washed five times in PBST. Brains were then incubated with 1:1,000 Alexa 568 anti-rabbit (Life Technologies) and 1:1,000 Alexa 633 anti-mouse (Life Technologies) antibodies for 4 h at room temperature before washing five times in PBST and coverslip mounting in Vectashield (Vector Labs). Images were taken at × 40 magnification on a Leica SP5 confocal microscope at 0.5 mm intervals and reassembled for display as maximum projections using Fiji56 (link).