1. Dissect embryos in cold PBS containing 2 mM EGTA. Day 9 embryos and older should have the brain punctured to allow solutions in and out.

2. Fix embryos in 10-15 ml of fresh, cold 4% paraformaldehyde/2 mM EGTA in PBS for two hours at 4°C with rocking.

3. Wash with cold PBT (PBS containing 0.1% Tween-20). Rock for 1 hour at 4°C. Transfer the embryos into 100% methanol, mix well, and store at -20°C.

4. Rock the embryos in 5:1 methanol/30% hydrogen peroxide for 1-2 hours at room temperature. The embryos can be stored in methanol at -20°C. Rehydrate the embryos through a graded series of methanol/CT (4:1, 1:1 and 1:4. CT is 10 mM Na Citrate pH 6.0, 0.1% Tween-20) at room temperature. Wash through three changes of CT.

5. Place the embryos in CT in a boiling water bath for 10 minutes. Try not to giggle.

6. Rock for 1 hour or more in MABT containing 2% Blocking Reagent at room temperature. (MABT is 100 mM maleic acid pH 7.5, 150 mM NaCl, 0.1% Tween-20. Blocking Reagent from Boehringer Mannheim is acidic. If dissolving in prepared MABT, adjust the pH back to 7.5 before use.)

7. Rock overnight at room temperature with primary antibody in MABT containing 2% Blocking Reagent.

8. Wash three times with MABT containing 2% Blocking Reagent, then 5 or 6 times with MABT, one hour each, at room temperature with rocking. Wash embryos 9 days and older in 10-15 ml.

9. Rock for 1 hour or more in MABT containing 2% Blocking Reagent.

10. Rock overnight at 4°C with secondary antibody (peroxidase conjugated) in MABT containing 2% Blocking Reagent.

11. Wash three times with MABT containing 2% Blocking Reagent, then 5 or 6 times with MABT, one hour each, at room temperature with rocking. Wash embryos 9 days and older in 10-15 ml.

12. For lower background, leave the embryos overnight at room temperature without rocking.

13. Wash in MABT, then incubate with 0.3 mg/ml DAB in MABT at room temperature for 20 minutes in reduced light. DAB is a carcinogen--handle with caution.

15. Wash the embryos with PBT. Clear the embryos in 1:1 glycerol/PBT, and then 4:1 glycerol/PBT containing 0.02% sodium azide. Store in the dark.