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		Materials and Methods Grafting The limbs used in this experiment were excised from 8 day old chick embryos, and the host embryos that received the grafted limbs were 10 days old. The grafted limbs were placed in Howard Ringer's solution (DB Lab, 2003) until they could be moved to the CAM of the host embryo. Thirty six host embryos were used in this experiment, each containing a single limb graft. Egg shells were demarcated in lead pencil to differentiate between limbs and wings, as it can be difficult to distinguish between the two during later development. The average survival rate for host chicks was 11/16, with the successful retrieval of about 18 limbs. Therefore, it helps to have a large population of host chicks to ensure a large enough sample of limbs in order to more accurately assess the occurrence of joint formation. The limb bud grafts were removed from the host eggs seven days later and placed in embryo ringers solution. Alcian Green Staining and Dehydration Excised limbs were then taken out of embryo ringers solution and fixed in 5% TCA for one hour at room temperature and rinsed twice with PBS solution. The limbs were then immersed in alcian green stain overnight. The alcian green stain was prepared according to the protocol listed on Developmental Biology Lab Website (DB Lab, 2003). In order to dehydrate the limbs after staining, limbs were induced over a period of four days with successively greater concentrations of lithium chloride at 70, 85, 95 and 100 per cent, immersing in each concentration overnight. After the dehydration process, limb buds were placed in methyl salicylate and stored in glass containers, as methyl salicylate will dissolve plastic. Embryos were photographed after twenty three hours. Results A) wing bud B) leg bud C) leg bud Figure 1. Control limbs were detached from fifteen day old chick embryos and stained for cartilage with alcian green. Arrows point to examples of fully formed joints, spaces created by cell death along rods of cartilage. A wing showed digit and elbow joints (A). The joints of the toe digits were clear in a chick foot (B1). The knee joint and toe joints were easily seen in a chick leg (B2). A B C D Figure 2. Fifteen day old limb grafts (eight day old limbs grafted for seven days) were extracted from seventeen day old hosts and stained for cartilage with alcian green. Arrows point to examples of fully formed joints, spaces created by cell death along rods of cartilage. Leg grafts showed somewhat regular patterning and discernable toe and knee joints (A and B). A leg graft showed an irregular patterning, but discernable joints (C). Another leg graft showed an irregular patterning (possibly a duplication of limbs) and discernable joints (D). A B Figure 3. Fifteen day old limb grafts (eight day old limbs grafted for seven days) were extracted from seventeen day old hosts and stained for cartilage with alcian green. Arrows point to examples of fully formed joints, spaces created by cell death along rods of cartilage. A wing had a somewhat regular patterning and discernable digit and elbow joints (A). Another wing had a somewhat regular patterning and somewhat discernable elbow joint (B). A B Figure 4. Fifteen day old limb grafts (eight day old limbs grafted for seven days) were extracted from seventeen day old hosts and stained for cartilage with alcian green. Both wing grafts showed irregular patterning and cartilage/bones were completely intact with no discernable breaks in cartilage/bone formation (A and B).   All limbs were extracted at day seven of experimentation when chick embryos and grafts were approximately fifteen days old. Limbs were stained for cartilage with alcian green and observed and photographed under a microscope. Discernable gaps along rods of cartilage/bone were noted. Green staining indicated cartilage, blue staining cartilage and bone, and no staining either gaps in cartilage or bone or fully formed bone. Control limbs detached from fifteen day old chick embryos were normally patterned, relatively fully formed wings and legs (Figure 1 A, B1, B2). No staining occurred in upper regions of the leg, and in discrete spaces between green or green-blue stained regions (Figure 1). Patterning and staining varied among grafts. Three of seven (43%) hind limb or leg grafts were patterned somewhat normally, that is, they looked like the control leg (Figure 2 A, B). The remaining four (57%) hind limb grafts were irregularly patterned, that is, did not look like the control leg (Figure 2 C, D). No staining occurred in discrete spaces between green or green-blue stained regions similar to the hind limb control (Figure 2 A, B, C, D) in all hind limb grafts. Most fore limb or wing grafts (seven of nine&emdash;78%) were patterned like the control wing (Figure 3 A, B and Figure 4 A, B). No staining occurred in discrete spaces between green or green-blue stained regions (like the fore limb control) in six of nine (66%) of our wing grafts (Figure 3 A, B). In one fore limb (11%) green-blue staining appeared hazy where discrete areas of no staining occurred in the control (Figure 4 A). Green-blue staining occurred in continuous rods with no discernable spaces along those rods in two of nine (22%) fore limb or wing grafts (Figure 4 B). These two fore limb grafts were among the seven grafts that were patterned like the control (Figure 4 B).
		J. N. White 5/13/04