Fly brains were dissected on ice and fixed overnight in 4% paraformaldehyde at 4°C. For immunoenzyme staining, brains were transferred to 0.3% hydrogen peroxide () in methanol for 10 min to quench endogenous peroxidase activity and treated with citrate antigen retrieval solution (pH 6.0) for 15 min at 95°C after washout of . After blocked 30 min with 5% normal goat serum, brains were incubated for 48 h at 4°C in primary antibodies and then for 24 h at 4°C with secondary antibodies. Next, brains were developed for 30 to 50 min in 0.4 mg/mL DAB (Sigma, St Louis, Missouri) containing 0.005% and sequentially dehydrated in 50, 70, 85, 95, and 100% alcohol, 100% alcohol-acetone (1:1), and 100% acetone (). After dehydration, brains were sequentially infiltrated in 50, 75, and 100% () Spurr resin (SPI, West Chester, Pennsylvania) for 30 min each, followed by fresh 100% Spurr resin overnight. Finally, brains were embedded in 100% Spurr solution and polymerized for 36 h at 60°C. For immunofluorescent staining, fly brains were absolved from and citrate treatment, and finally mounted in the glycerolbased Vectashield.