Notes

1. Egg albumen is bactericidal, so instruments need not be sterilized, but they should be thoroughly cleaned after use and may be dipped in 70% v/v ethanol and allowed to air-dry before use. On no account should instruments (other than tungsten needles) be flamed—it destroys the temper of the steel.

2. Some workers prefer to cut an incomplete circular aperture, tipping this back and out of the way (like a trapdoor) during manipulations, and then closing it and sealing with tape on completing the procedure. Keep the outside of the egg dry, since the tape will not stick to a wet surface.

3. Instead of storing the explanted notochords on ice, you may directly graft one into a readily prepared host. However, do not keep the grafts at room temperature for long as they will coil up and become intractable.

4. Do not inject the contrast medium until each egg is ready for grafting—it can disperse quite rapidly. You may have to test India ink of several suppliers for toxicity, since the best brand (Pelikan "Fount") is now hard to find.

5. To induce sclerotome ectopically, the procedure in Subheading 3.4., step 2 may be slightly varied. The paraxial mesoderm gains competence to respond to noto-chordal signals at the time of somite formation. In addition, newly formed somites remain labile with respect to their dorsoventral and mediolateral pattern. Therefore, it is not critical to use HH 9-10 embryos or operate at the level of the open neuropore. You may find it convenient to insert the graft at the level of the anterior segmental plate.

6. During operations make sure the needle is kept clean of adherent albumen and tissue by passing it quickly through the flame of a Bunsen burner. Keep the needle in a hot flame to remove and resharpen a bent tip.

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