- 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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