![IMAGE imgs/Lyuda01.gif](http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Frog/imgs/Lyuda01.gif)
![IMAGE imgs/Lyuda02.gif](http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Frog/imgs/Lyuda02.gif)
EMBRYO CELL AND TISSUE CULTURE TECHNIQUES Rearing solution: 10% Modified Steinberg's Solution. Operating solution:Full strength HBSt solution. |
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| ![IMAGE imgs/Lyuda03.gif](http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Frog/imgs/Lyuda03.gif)
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| 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.
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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. |
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| 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 HBStbeforeusing.
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Results:
The transplant adhered, and the eye formation took place. It was necessary to make sure that the transplant adheres to the flank of the albino, otherwise the wound will not heal, and the transplant will dissociate from the embryo. During the experiment the embryo continued its development up to the stage 36. Since at the stage 16 the cells in the eye forming region are already committed to become neural cells, the transplanted patch was
able to form independently, regardless of its new position.
![Lyuda4.jpg](http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Frog/img_frog/Lyuda4.jpg)