Texas red lysine fixable dextran

Six week old male BALB/cAnN‐nu mice were injected intravenously with 15 µg of sEVs or PBS as control. Twenty hours after sEV injection, mice were injected intravenously with Texas Red lysine‐fixable dextran (70 000 M_W, Thermo Fisher Scientific) at 100 mg kg⁻¹. After 3 h, mice were injected intravenously with 10 mg kg⁻¹ Alexa Fluor concanavalin A (Thermo Fisher Scientific). Ten minutes later, each mouse was anesthetized and perfused with PBS and followed by 4% paraformaldehyde in PBS. Lung tissues were excised and immersed in 30% sucrose in PBS overnight. Tissues were cryosectioned with 12 µm thickness. Tissue sections were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Thermo Fisher Scientific) and examined under confocal microscopy for vascular leakage indicated by Texas Red signal. Three random fields of each section were captured, and three sections per lung were examined. Images were processed and analyzed by ZEN software.

Male 6 week old BALB/cAnN‐nu mice were injected intravenously with 15 µg EVs or PBS as control. 20 h after EV injection, mice were injected intravenously with Texas Red lysine‐fixable dextran (70 000 MW, Thermo Fisher Scientific) at 100 mg kg⁻¹. After 3 h, mice were injected intravenously with Alexa Fluor concanavalin A (Thermo Fisher Scientific) at 10 mg kg⁻¹. 10 min later, each mouse was anesthetized and perfused with PBS and followed by 4% formaldehyde in PBS. Lung tissues were excised and immersed in 30% glucose in PBS overnight. Tissues were cryosectioned at 12 µm thickness. Tissue sections were stained with DAPI (Thermo Fisher Scientific) and examined under confocal microscopy for vascular leakage. Five random fields of each section were captured and three sections per lung were examined. Images were processed by ZEN software and the area of dextran was quantified by Image J software.