Angiospark 680

For intravital microscopy, only STZ-diabetic mice were used. First, the mice were anesthetized with isoflurane (0.8 l/min, 2.5%, Baxter Deutschland GmbH), and an abdominal body window was implanted for repeated kidney imaging as previously described (Schiessl et al., 2019 (link)).
The next day, mice were anesthetized with isoflurane (0.8 l/min, 1.5%), intubated, and catheterized into the lateral tail vein. To maintain body temperature during intravital microscopy, the mice were kept on a heating plate.
Image acquisition was performed using an upright Leica SP8 multiphoton laser scanning microscope with a 40x/1.1 NA water immersion objective at the Core Facility Cellular Imaging at the Technical University Dresden. Multiphoton imaging was performed with 860 nm laser excitation to visualize Angiospark680® (Perkin&Elmer, 30 µl), Hoechst 33,342 (Thermo Fischer, 50 µl of 2 mg/ml stock), and Lucifer Yellow (LY; Sigma-Aldrich).

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).