In Vivo Imaging of Extracellular Vesicles

1 µl of labeled EVs was injected into mouse dorsal ear dermis. After 30 min, the mouse was sacrificed and perfused with Ringer’s solution followed by zinc fixative (4.5 mM CaCl₂, 52 mM ZnCl₂, 32 mM Zn(CF₃COO)₂, 2 mM Tris, and 38 mM glycine, pH 6.5, 340 mOsm/liter). Whole mount staining of the ear was performed as described previously (Kilarski et al., 2013 (link)). Ears were cut and fixed for 24 h in zinc fixative and 1% Triton X-100. The dorsal skin was separated from the rest of the ear, washed in TBS, and blocked for 1 h in TBS 0.5% casein. The dorsal skin was incubated with anti-VE-cadherin antibody (550548; BD Biosciences) and anti-podoplanin antibody (AF3244; R&D Systems) for 24 h, washed in TBS 0.1% Tween, and incubated with secondary antibodies for 24 h. After washing in TBS Tween 0.1%, the tissue was dehydrated with 70% ethanol and 100% ethanol and cleared and mounted on a glass slide in 2:1 benzyl benzoate/benzyl alcohol solution. Fluorescence images were acquired with an Olympus IX2-DSU fluorescence microscope and a 63× lens. Image stacks were processed with ImageJ (National Institutes of Health).