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		Materials Needed/ Preparation Sheet 1. Sterile HEPES-buffered Modified Steinberg's Solution (HBSt) with gentamycin. 2. Axolotl neurulas (Hosts~Stage 25 to maximize survival; donors~Stage 15) 3. 60 mm Petri dishes: coated w/ 2% agarose in 1x HBSt 4. 70% Ethanol (EtOH) for sterilization of tools and pipettes 5. Forceps, tweezers, hair loops, eyebrow knives and Tungsten microscalpels, wide-mouthed pipets (for how to make eyebrow knives or Tungsten scalpels, pls. see References) Procedure 1. Choose several embryos to 'dejelly' (remove their jelly coats in order to manipulate them). First, sterilize all tools with 70 % EtOH, and then dip them into HBSt. Grasp the jelly coat of the selected embryo with a pair of forceps. Using a second pair of forceps, pull away and discard the jelly coat from the embryo. 2. Allow to sit for a few minutes so that the vitelline membrane swells. Remove the membrane around the embryo by gently plucking its surface with a pair of forceps. Once the forceps have snagged the membrane, use a second pair of forceps to pull it away from the embryo entirely. Repeat this for all of the selected embryos. 3. Choose two embryos, one as a donor and one as the host. Using a wide-mouthed pipet, transfer both of them to a Petri dish containing 1xHBSt and antibiotics. Locate the anterior end of the embryo. Rotate it so that its neural fold (which looks like a small groove running down the embryo's dorsal side) is facing up. This part of the embryo should contain the presumptive eye region. 4. Using an eyebrow knife or a tungsten needle, make very shallow cuts into the anterior portion of the embryo in the eye region (Figures 1 and 2). Figure 1. Anterior end of Axolotl neurula Figure 2. Axolotl neurula (side view) 5. Make four incisions to excise a very thin rectangular layer of tissue. Do this twice, once on each side of the anterior region, so that the chances of a successful graft increase. 6. Keep the graft tissues sterile until they are ready to be transplanted. 7. Using hair loops to move the embryos and graft tissues, position the albino host embryo with the neural folds to one side, so that its flank faces up. Locate the anterior end of this embryo. The incision should be made closer to the anterior end than to the posterior end, as this is likely to increase chances of a graft taking, since anterior cells are more likely to be competent to respond to eye-forming signals than posterior cells. However, do not make the incision in the head region, as this tissue is also eye precursor - then the experiment will not truly test whether the graft cells have been determined as eye cells or not. 7. Posterior to the head region, carefully make an incision a little bigger than the size of the graft tissue, running from the anterior end to the posterior end. Be careful not to cut too deep. Figure 3. Where to place the graft in a host Axolotl neurula 8. Transfer the grafts to the Petri dish containing the host embryo. Place one graft into the incision. Make sure it is firmly in the incision and not merely on top of it, so it will stay in place once the incision heals. The easiest way to do this is to simply push the graft into the incision with the blunt side of a tungsten needle. 9. Take photographs of the host embryos. 10. Come back over several days and take photographs to record and observe any grafts that did take. On the second day, transfer them to 50% HEPES rearing solution (the embryos should only be kept in the antibiotic solution as long as it requires them to heal). Remove any dead embryos from storage and discard; they will look as though they have 'exploded' or otherwise will be severely debilitated. On the third day, move the surviving embryos to 20% HEPES rearing solution.