Protocol
note: When the embryos are not being manipulated, keep them in a solution of HBSt containing antibiotics in order to keep the cell hydrated and to minimize the chance of infection.
1. Manually remove the jelly coating surrounding a stage fourteen Axolotl embryo using blunt foreceps.
2. Rinse an agarose Petri dish with an alliquot of the HBSt solution.
3. Place a prepared embryo into the operating dish containing HBSt with anitbiotics.
4. Maintain sterile surgical conditions through out the procedure by cleaning all tools in 70% ethanol and following surgical procedure.
5. Remove the membrane around the embryo by gently tearing with fine foreceps. Be careful not to crush embryo, and make sure to keep the embryo under the surface of the HBSt solution while removing the membrane. If done correctly, the embryo should slide out one side of the membrane.
6. Using an eyebrow knife, create a small ventral pocket in the right-hand side of the neural crest region of the recipient (regularly pigmented) embryo (Figure 2a). No removal of cells is necessary in the recipient.
7. Remove a section of the neural fold from the right side of the donor (albino) embryo. Use the eyebrow knife to make a thin, consistent incision in the lightly pigmented epidermis very close to the distal edge of the neural fold (Figure 2a). Continue to cut until the unpigmented cells beneath the surface are exposed.
8. Make an incision parallel to the initial incision along the inner edge of the right neural fold (Figure 2b).
9. Extract the section with two short cuts in order to harvest the rectangular cell sample (Figure 2c). If the excision is sucessful, the sample should include the whitish endodermal cells as well as a layer of the tan-colored ectoderm.
10. Using a surgical loop to manipulate the excised portion, transfer the neural fold segment from the albino donor to the regularly pigmented recipient. The implant should fit snugly within the incisio made in step 6.
11. Allow the embryos to heal. Then transfer both the donor and the host embryos into a sterile dish containing lower concentration HBSt by gradually reducing the concentration until 20% HBSt is acheived.
12. Allow development to continue until full grown axolotls are obtained.
13. Observe whether the grafting procedure was sucessful, and note any difference in the development of the grafted areas, particularly the pigment. Photograph the resulting embryos.