Fluorescence labeling and microscopy of adult Drosophila mushroom body:
1. Dissection: The brains are removed in cold Phosphate-buffered saline (1X PBS, pH 7.4).
2. Fixation: Tissues are fixed for 20 min in 4% paraformaldehyde (Merck, Germany) in PBST on an orbital shaker at room temperature.
3. Washing: Three quick (each 30 sec) and three long washing steps (each 15 min) and once for 2h with PBST on an orbital shaker at room temperature.
4. Blocking: 5% normal goat serum in PBST are used as a blocking solution for 30 min on an orbital shaker at room temperature.
5. Primary antibody: Brains were incubated for 2 nights in mouse anti-fasciclin II monoclonal antibody (Fas2; 1D4, Developmental Studies Hybridoma Bank, Iowa City, IA) diluted 1:20 in 5% NGS and at 4°C on an orbital shaker.
6. Washing: Brains are washed for 3×30 sec and 4×15 min with PBST at room temperature with orbital shaker.
7. Secondary antibody: Brains are incubated with Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Cat#A-11032, ThermoFisher Scientific) at a 1:200 dilution in 5% NGS and PBST for 24hr at room temperature on an orbital shaker in the dark.
8. Washing: Brains are washed for 3×30 sec and 4×15 min with PBST at room temperature with orbital shaker.
9. Mounting: Brains are placed on glass microscope slides and mounted in antifade mounting medium Vectashield® (Vector Laboratories, Burlingame, CA).
10. Slides are stored at 4°C in the dark until microscopy.

Points:
– Use 1X PBS, pH 7.4, 0.3% Triton X-100 (PBST) in step 2.
– Use 10X PBS, pH 7.4, 0.3% Triton X-100 (PBST) in steps 3 to 8.
– Keep the dish containing brains in 1X PBS on ice and fix them within one hour of dissection.
– Transfer the brains using a100 μl pipette.
– Rinse the empty pipette tips twice with PBST (prevent tissues from sticking to the plastic tips).