Grafting of Neural Tissue Pieces

1. Take a host egg of the desired stage, remove the sealing tape, and remoisten the embryo with a few drops of Ringer solution.

2. Using a tungsten needle, lift up the vitelline membrane, and cut a flap above the embryo; keep this to the minimum size required for access to the region of interest.

3. Using one tungsten needle make cuts around the tissue to be removed in order to receive the graft (Fig. 1). To do this, insert the needle to a depth of 100 pm or so, and pull gently upward and separate tissues. Then gradually deepen the cut with successive strokes of the needle (see Note 2). It is preferable to make the hole slightly bigger than the piece you intend to put into it.

4. Then either remove the tissue piece by hooking it out with the needle, or leave it in the egg. If you do the latter and you are using unlabeled donor tissue, you should be confident that you can tell the grafted tissue piece apart from the host tissue you have just removed.

5. Retrieve the neural tissue piece to be grafted from the Petri dish using a P20 pipetman and yellow tip set on 10-20 pL. If there are problems with the tissue getting stuck to the plastic pipet, you can coat the inside of the yellow tip with sterile serum before use.

6. Draw some fluid into the pipet before drawing up the tissue piece. Otherwise the tissue may hit the meniscus and disintegrate. Then transfer the piece into the egg as quickly as possible. Do this on low magnification, and try to track the piece with your eye to avoid losing it.

7. Maneuver the tissue piece into place adjacent to the grafting site using a tungsten needle (Fig. 1).

8. Orient the piece as required, and check for several seconds to see that it does not move out of position. It is possible to place small fragments of sterile drawn glass or other small objects on top of the neural tissue fragment to keep it in place, but this is not normally necessary (see Note 3).

9. Seal the egg with a piece of opaque egg tape (preferably stretchy). If you intend to incubate the embryo for more than a few days, extra Sellotape around the edges is also a good idea.

10. Eggs should be replaced in the incubator and left until the desired stage for harvesting (see Note 4). Opening up to check the stage of development and then resealing for further incubation is not recommended, and leads to certain death. When calculating the stage for harvesting, assume a day's delay in the development of embryos.

11. To open the eggs, cut a slightly bigger hole than before in the top of the egg using curved scissors.

12. Cut and lift the embryo out using spring scissors and forceps or a spatula, transfer to Ringer's solution, and wash off excess yolk before further processing.

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