Figure 4

Immunofluorescence staining of BL2B95 tumors on CAM. A: Immunofluorescence staining of tumor cryosections, merged picture of four channels. Cells were stained with DAPI (blue), anti-Prox1 (green), anti-HLA A,B,C (red), and anti-MEP21 (yellow). Prox1 stains nuclei of lymphatic endothelial cells, HLA A,B,C probes BL2B95 cells, and MEP21 stains blood vessels (bv). B: Green channel of A showing anti-Prox1 staining of nuclei of LECs. C: Yellow channel of A showing anti-MEP21 staining. Capillaries (arrows) and larger blood vessels (bv) are visible. D: Immunofluorescence staining of a whole mount specimen showing a region approximately 1 cm apart from the solid tumor. Merged picture of three channels. The sample was stained with DAPI (blue), anti-Prox1 (LECs, green), and anti-HLA A,B,C (tumor cells, red). Blood vessels appear as blue lines due to nucleated chick erythrocytes. Note specific localization of tumor cells in lymphatics. Insert: Higher magnification of boxed area. Scale bars (A-C) = 100 μm; Scale bar (D) = 100 μm.