1. Dissect brains in Drosophila Ringers solution.

2. Fix 20'' (1hr max) in a 0.5ml microfuge tube with 5 formaldehyde-PBS on ice.

3. Rinse 2-3X with PBS (carefully remove solutions with drawn out pipets).

4. Block 1h @ room temperature (RT) on nutator.

5. Incubate overnight @ 4oC in primary antibody diluted in PBT.

6. Wash in 4-6 changes of PNT over a 2hr period @ RT.

7. Incubate 1-2h in secondary antibody @ RT on nutator.

8. Wash in 4-6 changes PBT (15'' each).

9. Mount in 80 glycerol-PBS.

Drosohpila Ringers, per liter: 6.5g NaCl 0.14g KCl 0.2 g NaHCO3 0.12g CaCl2 0.01g NaH2PO4

10X PBS 1.3M NaCl 76.0 g NaCl 0.07M Na2HPO4 9.94g Na2HPO4 (MW=142) H2O to 1L 0.03M NaH2PO4 3.6 g NaH2PO4 (MW=120)

Formaldehyde-PBS = 5 Formaldehyde (stock is 37) in 1X PBS

PBT =1 BSA +0.1-0.5 Triton X-100 in 1X PBS

PNT = 2 appropriate serum + 0.1 Triton X-100 in 1X PBS