In vivo Visualization of Transplanted Vessels

In vivo visualization of transplanted vessels was performed with a TCS/SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Chamelon laser (Coherent, Santa Clara, CA, USA) as previously reported^{49 (link)}. During imaging, an animal’s head was immobilized using a custom-made cranial window holder. Mice were kept on a heated pad set to 37 °C, and anesthesia was maintained with isoflurane vaporizer and scavenger (Muromachi Kikai, Tokyo, Japan). In experiments to observe the area and growth rate of transplanted vessels, mice were observed 7, 14, 21, and 28 days after transplantation. In an experiment to observe the relationship with recipient vessels, mice were observed 21 days after transplantation and were injected retro-orbitally with 100 μL of AngioSPARK 680 (PerkinElmer, Waltham, MA, USA).