1. Recent improvements to this protocol, incorporated above, include the high concentration of nonspecific RNA in the hybridization buffer (500 pg/mL), and long posthybridization and postantibody washing steps.

2. It is essential to use high-purity formamide in the hybridization buffer and wash solutions A, B, and C. Fluka formamide gives consistent results.

3. After prehybridization (Subheading 3., step 15), embryos can be stored at -20°C for later use.

4. In contrast to some other protocols, we omit an RNase step in the posthybridization washes; in amphioxus, it can reduce signal without enhancing specificity. Different results may be found in other species.

5. In our experience, the most critical variant is the quality of the probe. After synthesis, probes must be checked by agarose electrophoresis to assess concentration and degradation; storage in 50% formamide (step 3) greatly improves probe stability and dissolution.

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