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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.
Fix embryos in 10-15 ml of fresh, cold 4% paraformaldehyde/2 mM EGTA in PBS for two hours at 4°C with rocking.
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.
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/PBT (4:1, 1:1 and 1:4) at room temperature. Wash through three changes of PBT.
Rock with 10 µg Proteinase K in PBT for 7 minutes at 37°C. Wash twice with PBT and immediately refix for 20 minutes in 4% paraformaldehyde/0.1% glutaraldehyde in PBS for 20 minutes at room temperature. Wash three times in PBT.
Wash twice with 2 N HCl containing 0.1% Tween-20. Rock for 1 hour in the same. Wash three times with MABT.
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.)
Rock overnight at room temperature with primary antibody in MABT containing 2% Blocking Reagent.
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.
Rock for 1 hour or more in MABT containing 2% Blocking Reagent.
Rock overnight at 4°C with secondary antibody (peroxidase conjugated) in MABT containing 2% Blocking Reagent.
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.
For lower background, leave the embryos overnight at room temperature without rocking.
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.
Develop color by incubating with 0.3 mg/ml DAB and 0.03% H2O2 in MABT. Rock for 5 to 30 minutes in reduced light.
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.

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Whole Mount BrdU Staining

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