Nditions as the melanoma cells [16]. Cells were injected into the lumen of the neural tube by entering caudally at the site of the tail bud to prevent tissue damage. Injections were performed at stages 12?3 HH, during or shortly after closure of the neural tube (Figure 2B). In the case of GFP-labeled B16-F1 cells, GFP epifluorescence was used to demonstrate the site-specific transplantation result (Figure 2C). Embryos were further incubated for 48h; GFP epifluorescenceillustrated dorso-ventrally migrating melanoma cells in lateral view of the embryo (Figure 2D). At stage 20 HH the embryonic optic cup is localized at the surface of the chorioallantoic membrane and easily recognized because the pigment epithelium has just developed. For transplantation into the optic cup, eggs were fenestrated after 72?0 h of incubation (corresponding to stage 19?0 HH). B16-F1 aggregates or melanocyte aggregates (untreated, bone morphogenetic protein (BMP)-2 pre-treated or nodal pre-treated; n = 7 embryos per group) were transplanted into the optic cup (Figures 2E, F and Table 1), entering at the site of the choroid fissure of the optic cup (pointed out in Figure 2H). In some cases, local capillary bleeding occurred, which usually stopped within 1?2 min without disrupting embryo development. For better visibility and documentation purposes, B16-F1 melanoma cell aggregates were stained with nile blue sulphate before transplantation (Bayer, Leverkusen, Germany). After transplantation, the aggregates remained at the site of transplantation and were documented. Eggs were sealed with adhesive tape and further incubated for 72 h (Figure 2G). For transplantation into the brain ventricles, the capillary was entered into the embryo cranially at the most caudal site of the rhombencephalon (Figures 2I, J), and embryos were incubated for additional 48 or 96 h (Figures 2 K, L). 95 of the embryos that were transplanted into the neural tube, and 80 of the embryos that were transplanted into the brain ventricles or into the optic cup survived the transplantation procedure and the following reincubation time ranging between 24 and 96 h.The Chick Embryo in Melanoma ResearchFigure 3. Histology, immunohistochemistry 
and in situ Autophagy hybridization of the chick embryos. (A) Schematic drawing depicting ventral and medial neural crest migration pathways. n.c. neural crest; n.t. neural tube; s.t. sympathetic trunk. (B) Chick embryo 24 h after transplantation of SKMel28 melanoma cells into the neural 15755315 tube. Melanoma cells (visualized by HMB45 immunoreactivity) spontaneously resuming neural crest migration have a stretched, mesenchymal-like morphology (arrows). (C) At the site of destination along the ventral migration pathway (para-aortic sympathetic ganglia) melanoma cells undergo apoptosis, visualized by TUNEL staining. (D,E) Chick embryo 24 h after transplantation of benign primary human melanocytes into the neural tube. Melanocytes (showing a compact, epithelial-like morphology) are encountered only in the lumen of the neural tube and, in part, integrated into the roof plate with no neural crest migration. (F) Melan A immunoreactivity confirms the melanocytic origin of the cells. (G) Schematic drawing of chick embryo 72 h after transplantation of B16-F1 melanoma cells into the optic cup. (H) Histological correlate of schematic drawing. Already in H E staining the transplanted, invasively migrating melanoma cells are visible (arrows). (I) Single melanoma cells (identified by HMB45.
