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		Procedure: Graft technique 1. Follow previous protocol for preparation of microtools and operating dishes, dejellying, and demembranation of stage 15 recipient embryos (Figure 1 A, below) and stage 20 donor embryos (Figure 1 B). 2. Position donor embryo on lateral side and recipient embryo on dorsal side (neural crest-side down) exposing ventral side in separate wells of operating dish. 3. Hold donor embryo with hair loop instrument and use Tungsten microscalpel to remove small chunk of tissue from lateral, gill-forming surface. CAUTION: Do not remove a chunk of neural tube. These have the tendency to adhere strongly to themselves and will therefore not heal well in the recipient embryo. 4. Hold recipient embryo ventral side up with hair loop and pick a small slit down the midline of the embryo with eyebrow microscalpel or tungsten microscalpel (Figure 2 A). CAUTION: Be gentler than when cutting the donor gill chunk. Do not slice through the entire embryo and do not cause inner cells to spew out of embryo! 5. Imbed donor gill-forming piece in the incision on the recipient embryo. Pat down and ensure its position with eyebrow or tungsten microscalpel. 6. Leave one recipient embryo with only the incision but no graft, and one embryo uncut as controls. 7. Observe the development of the embryos until heart forms. 8. As described previously, the high Ca2+ concentration of the HBSt enhances healing by saturating the Ca2+ binding sites of cadherin molecules, but a high salt concentration also causes developmental abnormalities. Therefore, after embryos heal from surgery, gently replace 100% HBSt with 50%, then 20% HBSt. Change solution concentrations after 12 hrs. 9. Collect pictures and movies daily using a microscope equipped with a still camera and recording cameras. A need spaceB Figure 1. Dejellied test embryos. (A) Stage 15, early neurula II recipient embryo. Clear vitelline envelope is swollen and visible after soaking in 100% HBSt. (B) Dejellied, demembranated, stage 20, late neurula IV donor embryo. Donor grafts were taken from the gill-forming region of a stage 20, late neurula IV, shown by arrow.     Need space Figure 2. Recipient embryos. (A) Stage 16 recipient embryo with incisions down midline of ventral side. Actual incisions were larger than this photograph portrays. (B) Recipient embryo with graft in incision. Dark donor tissue sits in lighter area of incision. Movie of the dejellying, demembranation, and grafting procedure. Click here to open a laboratory handout of the Protocol and Prep sheet