Immunostaining Protocol for Drosophila Brains

Immunostaining was performed as previously described in [11 (link)]. Briefly, fly heads were fully dissected and fixed in 400 µL of 4% paraformaldehyde (PFA), 0.3% Triton X-100 for 20 min on ice. Once fixation was completed, three quick washes followed by three 20-min washes were performed in PBS, 0.3% Triton X-100. Blocking was achieved with 1 h incubation in 5% normal goat serum (NGS), PBS, 0.3% Triton X-100 at room temperature on a rocking platform. Brains were incubated with the primary antibodies for two nights at 4°C on a rocking platform. Brains were then washed and incubated with the secondary antibodies overnight at 4°C. Vectashield (Vector Laboratories) was used as slide mounting medium. Antibodies used in this study and dilutions were as follows: Mouse anti-nc82 (mouse, Developmental Studies Hybridoma Bank, 1:100), Rabbit anti-GFP (G10362, Invitrogen. 1:500), Goat anti-Rabbit IgG Alexa Fluor 488 1:200 (A11008, Thermo Fisher, 1:200) and Goat anti-mouse IgG-Alexa Fluor 633 1:200 (mouse, A21052, Thermo Fisher, 1:200).