Figure 4: Immunohistochemical analyses of the primary tumors (mouse ears) after 2 and 4 weeks of sponge implantation.

Control sponges devoid of tumor cells (control) and sponges populated with 1.5 × 10⁵ or 2 × 10⁵ B16F10Luc+ tumor cells were implanted in C57BL/6J mouse ears for 2 (a) or 4 (b) weeks. Lymphatic initial vessels (LYVE-1 positivity, blue staining) and tumor cells (tyrosinase expression, pink staining) were detected by immunohistochemistry, in control ears (sponges soaked with serum-free DMEM without tumor cells) and in ear-bearing primary tumors. Representative pictures are belonging to the central area of the ear sponge. Lower panels in “a” and “b” represent a higher magnification of the region delineated by the square in the upper pictures. Black arrows indicate some representative lymphatic vessels and the dotted black lines in “b” delineate the well-established tumor areas. ESL and ISL indicate the external and internal skin layers in mouse ears, respectively. Asterisk indicates the cartilage in mouse ears. Scale bars represent 500 and 100 μm in the low and high magnification pictures, respectively.