1. For grafting of very young embryos (younger than stage 9), it may be desirable to avoid opening the eggs until immediately before grafting, since dehydration is a major cause of mortality. Replacing the cut piece of shell over the hole and securing with tape may also improve survival rates.
2. Always keep the motion of the needle toward you, rather than down into the egg, since this guards against damage to the embryo. Use more smaller cuts rather than few larger ones to free the region from its surrounding tissues. For rhombomere grafting, this involves cuts at the rostral and caudal ends, the lateral edge where the neural tube abuts the mesenchyme, in the dorsal midline, and the ventral midline (where the ventral neural tube abuts the floor plate).
3. If you have been struggling for many minutes with the graft, you may notice that the surface of the embryo has dried out, and needs the addition of Ringer solution. If so, do this very carefully to avoid dislodging the graft.
4. The major problem of all neural grafting experiments is the low survival rates of the grafted embryos. Even if the operations have been performed flawlessly, mortality can be about 50% after several days, falling to as low as 10% after a week or so. The causes of this are unclear, but the major factors are likely to be dehydration of the embryo and/or contamination. Dehydration can be prevented by addition of Ringer solution, performing the operation in the shortest possible time, and making sure the egg is well sealed at the end. Contamination should not be a problem provided sterile plastics, solutions, and tools are used throughout, and the only tool used inside the egg is a tungsten needle, which is frequently flamed. Excessive damage to the embryo, surrounding membranes and yolk and the developing vasculature, are also obvious problems, which should diminish with practice. Low survival rates produce the related problem that sample sizes in any one experimental category are very small. It may take a long time to obtain data that are amenable to statistical analysis. Furthermore, experimental results that are obtained within these categories may sometimes be heterogeneous owing to differences in the grafting procedure. For this reason, is may be desirable to label the grafted tissue in some way (see Subheading 1.).
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