To further confirm tumor invasion into lymphatic vessels, double-fluorescence immunohistochemical analysis against podoplanin and Melan-A was applied in paraffin-embedded sections. After preincubation of the slides with normal goat serum, 10% (Dako) in typical order, podoplanin and Melan-A immunoreactivity was visualized by corresponding Alexa568 goat anti-mouse and Alexa488 goat anti–rabbit IgG-tagged antisera (1:1000) (Invitrogen, Karlsruhe, Germany), respectively. Slides were covered with Tris-buffered saline–glycerol mounting medium (1:1 at pH 8.6). Negative controls were performed by omission of the primary antibodies during incubation and resulted in no staining. Furthermore, the tissue was treated with a commercially available negative control reagent (Dako), which resulted in no staining. To avoid misinterpretation in the masked studies, color coding (podoplanin, red; Melan-A, green) was maintained throughout the experiments and the report. For documentation, an inverted fluorescence microscope was used (Axio Observer Z1; Carl Zeiss Micro Imaging, Göttingen, Germany) with ×20 dry and ×40 and ×63 oil immersion objective lenses (numeric apertures of 0.8, 1.3, and 1.4, respectively) connected to a confocal laser scanning unit (LSM 710; Zeiss). Sections were imaged using the single optical section mode with the appropriate filter settings for Alexa568 (excitation, 568 nm; Zeiss filter block, 43; channel 1, coded red) and Alexa488 (excitation, 488 nm; Zeiss filter block, 38; channel 2, coded green).