Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and a broad range of pathological associations. A major technical limitation to understanding the role of EVs in normal and diseased specimens has been the inability to visualize the spatial localization of EVs in tissue microenvironments. Here, we use bovine ocular tissue, the vitreous humor, as a model system to study EV imaging. We show that mammalian tissues crosslinked with conventional formaldehyde solutions result in significant EV loss, with subsequent reduced or negative EV signals; however, EV escape can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) that permanently holds these nano-sized particles and allows for visualization of EVs in normal and cancer tissues in situ.