Immunoreactivity) form a tumor, and single melanoma cells invade the choroid of the optic cup (arrows). (J) Chick embryo 96 h after transplantation of human metastatic melanoma cells into the brain SC66 biological activity vesicle at the hindbrain (rhombencephalon). The cells 22948146 form a large tumor in the dorsal neuroepithelium with (K) single HMB45 positive cells infiltrating the surrounding brain tissues. (L) MIB1 immunohistochemistry (proliferation marker not cross-reacting with chick cells) identifies melanoma cells during haematogenous spreading in blood vessels among host erythrocytes and lymphocytes, and in the surrounding neural tissue. doi:10.1371/journal.pone.0053970.gFixation of Embryos and Paraffin EmbeddingAt the end of the incubation period, embryos were removed from the eggs using forceps and bent scissors (Moria, Antony, France). Embryos that had received transplantations into the optic cup were decapitated. Entire embryos and embryo heads were fixed in 4 buffered paraformaldehyde for 12?4 h depending on the size of the embryo and were transferred into tissue cassettes (RotilaboH Macro, Carl Roth, Karlsruhe, Germany). After rinsing with water, samples were dehydrated with ethanol, treated with xylene and embedded in paraplast in a routine histology embedding automat. The final casting in the paraffin block iscrucial for future histological evaluation and therefore was performed in a similar manner in all embryos. It was determined, whether Solvent Yellow 14 site transverse or longitudinal serial sections of the site of transplantation yielded the best results. In the presented cases, transverse sections were chosen in most cases to get a full overview of the embryo permitting a depiction 
of both the medial and lateral neural crest cell pathways.Species Specific MarkersA major challenge is to identify single migrating melanoma cells among normal chick embryo cells. In the early chick embryo weThe Chick Embryo in Melanoma ResearchFigure 4. Pre-treatment with the TGFbeta family members BMP-2 or nodal induces invasive migration of human melanocytes in the optic cup. Untreated, BMP-2 or nodal pre-treated melanocytes were injected into the optic cup of the chick embryo (stage 20 HH). After 72 h of further incubation, the embryos were analyzed for tumor growth and invasion. Untreated melanocytes formed loosely aggregated tumors adjacent to the hyaloid vessels (left image in upper row), in the developing vitreous body and behind the lens (right image in upper row) without invasion. The BMP-2 and 15755315 nodal groups formed tumors in similar locations. In the BMP-2 group single melanocytes invaded the lens epithelium (insert in left image in middle row), the retina, the hyaloid vessels, and the choroid (right image in middle row; arrows pointing at melanocytes). In the nodal group single melanocytes invaded the choroid (lower row, arrows in right image) and the hyaloid vessels. doi:10.1371/journal.pone.0053970.gused immunohistochemistry of melanocyte and melanoma cell specific markers HMB45 and Melan-A. Since in the early chick embryo neural crest cells have not yet differentiated, they do not express markers of the melanocyte cell lineage. This changes at E6 during emigration of neural crest cells on the lateral pathway [15]. Now the melanocyte precursors of the chick embryo also become HMB45 positive. As absolutely specific marker, in situ hybridization with the species-specific DNA sequences Alu for human and L1 for mouse cells can be performed [21]. In contrast to the mouse, there is.Immunoreactivity) form a tumor, and single melanoma cells invade the choroid of the optic cup (arrows). (J) Chick embryo 96 h after transplantation of human metastatic melanoma cells into the brain vesicle at the hindbrain (rhombencephalon). The cells 22948146 form a large tumor in the dorsal neuroepithelium with (K) single HMB45 positive cells infiltrating the surrounding brain tissues. (L) MIB1 immunohistochemistry (proliferation marker not cross-reacting with chick cells) identifies melanoma cells during haematogenous spreading in blood vessels among host erythrocytes and lymphocytes, and in the surrounding neural tissue. doi:10.1371/journal.pone.0053970.gFixation of Embryos and Paraffin EmbeddingAt the end of the incubation period, embryos were removed from the eggs using forceps and bent scissors (Moria, Antony, France). Embryos that had received transplantations into the optic cup were decapitated. Entire embryos and embryo heads were fixed in 4 
buffered paraformaldehyde for 12?4 h depending on the size of the embryo and were transferred into tissue cassettes (RotilaboH Macro, Carl Roth, Karlsruhe, Germany). After rinsing with water, samples were dehydrated with ethanol, treated with xylene and embedded in paraplast in a routine histology embedding automat. The final casting in the paraffin block iscrucial for future histological evaluation and therefore was performed in a similar manner in all embryos. It was determined, whether transverse or longitudinal serial sections of the site of transplantation yielded the best results. In the presented cases, transverse sections were chosen in most cases to get a full overview of the embryo permitting a depiction of both the medial and lateral neural crest cell pathways.Species Specific MarkersA major challenge is to identify single migrating melanoma cells among normal chick embryo cells. In the early chick embryo weThe Chick Embryo in Melanoma ResearchFigure 4. Pre-treatment with the TGFbeta family members BMP-2 or nodal induces invasive migration of human melanocytes in the optic cup. Untreated, BMP-2 or nodal pre-treated melanocytes were injected into the optic cup of the chick embryo (stage 20 HH). After 72 h of further incubation, the embryos were analyzed for tumor growth and invasion. Untreated melanocytes formed loosely aggregated tumors adjacent to the hyaloid vessels (left image in upper row), in the developing vitreous body and behind the lens (right image in upper row) without invasion. The BMP-2 and 15755315 nodal groups formed tumors in similar locations. In the BMP-2 group single melanocytes invaded the lens epithelium (insert in left image in middle row), the retina, the hyaloid vessels, and the choroid (right image in middle row; arrows pointing at melanocytes). In the nodal group single melanocytes invaded the choroid (lower row, arrows in right image) and the hyaloid vessels. doi:10.1371/journal.pone.0053970.gused immunohistochemistry of melanocyte and melanoma cell specific markers HMB45 and Melan-A. Since in the early chick embryo neural crest cells have not yet differentiated, they do not express markers of the melanocyte cell lineage. This changes at E6 during emigration of neural crest cells on the lateral pathway [15]. Now the melanocyte precursors of the chick embryo also become HMB45 positive. As absolutely specific marker, in situ hybridization with the species-specific DNA sequences Alu for human and L1 for mouse cells can be performed [21]. In contrast to the mouse, there is.
