Grafting Tissues

The easiest method of grafting from embryo to embryo is to use a clay-bottomed dish. A hole the size of the embryo is made in black modeling clay with a ball tip, formed at the end of a disposable pipet by holding it in a flame until the correct size molten ball is formed. The host embryo is placed in the hole, graft site uppermost, if possible. If the embryo rotates, small nibs of clay can be pushed against it with forceps, holding it in position. The donor embryo is placed alongside the host, also in a depression (necessary only if one wants to keep the same levels of both embryos in focus at higher magnifications). A graft is cut out of the donor and a graft site cut in the host; which operation is done first depends on which is the most troublesome to do, and which preparation gives the most trouble when left alone for a time. Speed is crucial. If left alone too long, the graft will curl, and the hole made to accept it will first gape and then heal. The boundaries of graft and graft site should be closely matched, since healing will occur fastest under these conditions. If the graft is slightly larger than the site, it is important to tuck the edges of the graft into the graft site. If the edges of the graft overlap the host epithelium, healing will be delayed. The graft epithelium will turn back on itself and ultimately find the edge of the host epithelium, but this will take a while. In order to hold the graft in place, forceps are used to push up two long ridges of clay on each side of the embryo, some distance (2-4 mm) away. A small coverslip fragment, preferably a precut rectangle, is bridged across the two ridges, above the embryo. The coverslip is pushed down on the graft with forceps tips, taking care to align the surface of the glass exactly perpendicular to the graft surface, pushing it directly into the graft site. If the embryo or graft moves one way or the other as pressure is applied, the glass bridge can be tilted to counter these movements. Healing should occur in 15-20 min at which time the coverslip is removed and embryo is removed from the clay. Most batches of clay appear to be somewhat deleterious to the embryo over long periods, although embryos from many spawnings will develop normally to advanced stages on clay. We prefer black clay for contrast, but other colors can be used according to the experimenter's taste. Some colors appear to affect the embryos more than others.

The method described above is the easiest, but not necessarily the best way to graft tissues. With practice, one can make grafts between free, unconstrained embryos lying in a dish. This is the least disruptive and yields the best results, if done correctly. It is important that the edges of grafts are mated precisely. The grafts should be relatively small, and they must stick in the graft site without external pressure, all of which require much practice.

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