Pretreatments Prior to Hybridization

1. Rehydrate the embryos through a methanol/PBT series (75:25%, 50:50%, 25:75% methanol:PBT) finishing with three washes of PBT (see Note 16).

2. Fix the embryos with 4% paraformaldehyde in PBT for 20 min at room temperature. This second fixation helps prevent the embryos from falling apart.

3. Wash twice for 5 min in PBT.

4. Add 0.2-0.4 mL of hybridization buffer, 50-65% formamide, 5X SSC, 500 ^g/mL yeast RNA, 0.1% Tween-20, 50 |Jg/mL heparin, and 1 M citric acid to pH 6.0. The volume used depends on the number and size of embryos.

5. Incubate for 5 min, and then add fresh hybridization solution and incubate at 65-70°C for a minimum of 2 h.

6. The embryos are now safe from RNases.

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