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IMMUNOHISTOCHEMISTRY ON Drosophila BRAINS

Caution! In converting these protocols to HTML, some units have been scrambled.
Specifically, microliters (ul) often appear as milliliters (ml). Think first!

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