Manipulation

1. Embryos that are 8.5 d old are manipulated in a Petri dish in the same way as described in Subheading 3.3.4., steps 1-4.

2. Orient the embryo using fine watchmaker forceps.

3. Hold the embryo by the yolk sac with the holding pipet by applying negative pressure to the micrometer syringe. The embryo is positioned so that a clear silhouette of the cranial neural fold is visible.

4. Insert the injection/grafting pipet through the yolk sac, and then the amnion into the amniotic cavity.

5. Push the pipet gently into the cranial neural fold or further into the cranial mesoderm.

6. Expel the cell clumps or dye by applying a positive pressure to the de Fonbrune syringe. Expulsion of the cell clumps is accompanied by the simultaneous withdrawal of the pipet.

7. For dye delivery, the pipet remains in position until the dye front has stopped moving toward the tip.

8. Returned the embryo to culture (Subheading 1.).

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