Method

1. Follow steps 1-8 of the method in Subheading 2.2.2. to place a chick embryo in modified New (1955) culture as if to receive a graft.

2. Use fine needles to cut out the node, involving the whole thickness of the embryo but being very careful to avoid damaging the vitelline membrane underneath. Even a small hole will cause leakage of albumen and prevent healing, or even displace the graft. It is best to work in steps: first make a very superficial cut of the shape required. Then deepen the cuts, a little at a time, until the node is finally freed all around.

3. It is of advantage to mark one edge of the node with fine carbon (e.g., pencil lead shavings) or carmine (Sigma C1022) particles, using a fine needle. This allows the orientation of the node to be controlled through subsequent manipulations.

4. Manoeuver the excised node, using the fine needles, to the desired orientation, again taking care not to damage the vitelline membrane that is now exposed.

5. Still observing under the microscope, carefully and slowly withdraw as much saline as possible from inside and outside the ring, as described for grafting Hensen's nodes above. In this experiment, where the manipulated node is not secured under a flap of tissue, it is much easier to lose it while sucking off the fluid. If necessary, replace it in position as required, using the fine needles.

6. Once all the fluid has been removed, use the fine capillary and mouth tube (if necessary) to remove all remaining fluid from the site of the graft. It is likely that the excised piece will appear to have shrunk. Sucking the fluid off in this way will close the gap and "knit" the pieces together, if performed with care.

7. Now slide the ring from the watch glass and set up the culture as described in Subheading 2.2.3., items 7-10. Finally, if necessary, suck off some more fluid with the mouth capillary assembly to ensure that the graft site is totally dry and appears closed.

8. Before placing in the incubator at 38°C, it is advantageous to keep the operated embryo at room temperature (in the sealed Petri dish) for 2-3 h. After this, place it at 30°C for 3-5 h. Finally, transfer the dishes to an incubator at 38°C. These periods at lower temperature will help the healing process as described above.

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