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