Figure 4.

Podoplanin and VEGF-C staining in CCR7-B16 tumors. CCR7-B16 or pLNCX2-B16 tumors were harvested 20 days after tumor cell inoculation into ears of mice. Anti-podoplanin antibody (green; a, b) and anti-VEGF-C antibody (red; b) were used for immunofluorescence and immunohistochemical staining (for quantification of lymphatic vessels). Four random high-power fields (HPF), which contained lymphatic vessels (podoplanin-positive structures with vessel morphology) or VEGF-C-expressing cells in each section, were selected. The numbers of the lymphatic vessels and of the VEGF-C-expressing cells were counted, and representative results of two tumor samples are shown. Two sections were stained from each tumor for VEGF-C (b). CCR7-B16 or pLNCX2-B16 tumors were harvested 20 days after tumor cell inoculation into footpads of C57BL6 mice. Equal-sized tumors were collected and homogenized. CD45⁺ cells were sorted from tumor suspensions using FACSAria II and were attached to a slide glass using Shandon Cytospin 3 Centrifuge. Sections were stained using anti-VEGF-C antibody. Three HPFs, which contained VEGF-C-expressing cells, in each section were selected and VEGF-C-expressing cells were counted (c). Scale bars, 100 μm.