Stimulation of lymphatic and blood vessel growth in vivo. a–c Preparation of gelatin sponges for lymphangiogenic assay. a Small pieces of gelatin sponge are detailed with a puncher before to be soaked with tumor cells or a compound. Prepared sponges are inserted in mouse ear between the two skin layers. b, c Immunohistochemical analysis of sponges (b) and sentinel lymph node (c) resected from mouse with control sponge (PBS or medium without growth factor) or from mouse with sponge imbedded with growth factor. Lyve-1 (lymphatic endothelial cell marker) is stained in green, and CD-31 (blood endothelial cell marker) is stained in red. Sponges soaked with growth factor showed higher lymphangiogenesis and angiogenesis compared to control sponge. Scale bar in b, c—250 μm. d–f Preparation of AAV and transduction of skeletal muscle. d Schematic representation of different VEGF-C and VEGF-D isoforms, produced by step-by-step proteolysis, and general AAV production and usage protocol. e Immunohistochemical analysis of t.a. muscle transduced with AAV8 encoding VEGF-C-ΔNΔC or VEGF-D-ΔNΔC. Tibialis anterior muscles of C57BL/6 J male mice (8 weeks old) were injected with 109 AAV8 particles in 30 μl of PBS, and the mice were euthanized 2 weeks later. T.a. muscle samples were isolated and analyzed immunohistochemically for the indicated markers. HSA human serum albumin. f Analysis of the functionality of lymphatic vessels. Lectin (from Lycopersicon esculentum), conjugated with FITC (FITC-lectin) was injected to the distal part of t.a. muscle. After 45 min, the mice were euthanized and t.a. muscle was isolated, fixed, and stained for Prox1. Lectin is visualized by FITC. Scale bar in e, f—50 μm
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