TransplantationoftheeyeformingregionofAmphibianneurulaintotheflank

EMBRYO CELL AND TISSUE CULTURE TECHNIQUES

Rearing solution: 10% Modified Steinberg's Solution. Operating solution:Full strength HBSt solution.

Microsurgery Preparation

1)Place two embryos in the 60 mm petri dish containing Operating solution with antibiotics.
2)Using tweezers and forceps dejelly the embryo and remove the membrane around it.
3)Prepare 2% agarose-coated operating dishes in 1X HBSt (+/- powdered charcoal for
contrast), rinse with HBSt that contains antibiotics.
4)Place embryos into operating dish containing 1X HBSt and antibiotics. Use hair loops to
transfer the embryos. The donor and recipient embryos should be operated on separate
dishes. Place the donor on apetri dish with clear agarose gel, and the albino recipient
on an agarose powdered with charcoal.

Removal of the Eye Forming region:
Modified from Hamburger, V. 1960. A manual of Experimental Biology. The University of
Chicago Press, pp.107-109.

1)Position the donor embryo so that the anterior part of the neural fold is crearly visible.
2) Using eye-brow knives make very shallow cuts in the anterior portion of the embryo.
3)After the cuts are made carefully, not to tair the entire embryo apart, take off a very thin layer of the tissue.

Preparation of Albino Recipient Embryo:

1)Place the albino embryos with the flank afrea facing up.
2)Make an incision starting from the anterior to the posterior of the embryo.
3)Carefully transfer the grafts to the petri dish containing the albino embryo.
4)Place the graft into the flank cut of the albino. Make sure it stays in place so that the
wound can heal.
5) Observe the embryos after a few days to see whether the grafts have developed into an
eye. Take digital images of the grafted embryos.

 ^{Use clean technique; dip tools and pipettes in 70% EtOH and then into sterile HBSt}beforeusing.