1. All steps are performed at room temperature for 5 min with rocking or slow rotation unless otherwise stated (see Note 1). Times are increased to for older embryos (>embryonic day [E] 5.5 chicks or >E11 mice). The procedure as described below is suitable for avian embryos up about stage 26 (E5.5) chicks, E11.5 mice, and zebrafish at all stages. A more suitable protocol for Xenopus embryos is presented elsewhere in this volume.

2. To avoid damage, leave a small volume of liquid over embryos between each step.

3. Between fixation (Subheading 3.2., step 2) and washing after hybridization (Subheading 3.5., step 1), there must be no chance of contamination with RNases. Wear gloves and use disposable plastics instead of glassware where possible. Prepare all solutions with DEPC-treated double-distilled water.

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