Detection of Bound Probe

1. Incubate the embryos in 1 : 100 FITC-avidin D in 2X SSC, 0.1% Tween-20, 100 |g/mL RNase A overnight at 4°C on a rotating wheel (see Notes 3, 12, and 13). In this and the following steps, keep the tube covered with aluminum foil to prevent exposure to light.

2. Wash the embryos twice in 2X SSC, 0.1% Tween-20 for 20 min at room temperature.

3. Wash the embryos once in PBS for 5 min.

4. Incubate the embryos in 1 : 1000 propidium iodide solution in PBS for 5 min.

5. Rinse the embryos in PBS for 5 min.

6. Mount the embryos in Vectashield or similar mountant.

In case of weak FISH signals, see Notes 13 and 15.

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