Non-reversible tissue fixation retains extracellular vesicles for in situ imaging

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues. Tissue fixation with formaldehyde and a water-soluble carbodiimide crosslinker (EDC) leads to retention of extracellular vesicles within tissues and allows for reliable extracellular vesicle imaging for semiquantitative imaging applications.