Supplementary Figure 1: Extracellular vesicles (EVs) escape from formalin-fixed bovine vitreous tissues and are retained with formalin fixation and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide crosslinking.

a, Graphical representation of the size and concentration of EVs isolated from fresh bovine vitreous humor. The mean (black line) ± standard error (red bars) concentration of EVs according to EV diameter, based on nanoparticle tracking analysis (NTA) is presented. b, Schematic diagram showing formalin-fixed vitreous (Vit) tissue immersed in wash buffer (supernatant) and heated to 37ºC results in escape of EVs (arrowhead, red circles) and vitreous collagen (C, closed arrow) into the supernatant (wash buffer). c, Schematic diagram shows that the wash buffer was collected and the ultrastructural morphology characterized with transmission electron microscopy (TEM) or the concentration of EVs present in the wash buffer was determined by NTA. d, Representative TEM photomicrograph of supernatant collected from formalin-fixed bovine vitreous tissue after incubation at 37ºC and uranyl acetate (UA) and lead citrate staining shows evidence of collagen strands (C, closed arrow) and numerous EVs (arrowhead). e, Representative TEM photomicrograph of supernatant collected from formalin-fixed bovine vitreous tissue after incubation at room temperature (23ºC) shows evidence of EVs in the wash buffer (arrowhead). f, Representative TEM photomicrograph of supernatant collected from bovine-fixed vitreous tissue kept at 4 ºC reveals few collagen strands (C, closed arrow), and no EVs were detected on the photograph. g, Schematic diagram showing formalin–EDC-fixed vitreous tissue (Vit) immersed in wash buffer and heated to 37ºC resulted in retention of EVs (arrowhead, red circles) in the tissue, with no loss of EVs and minimal loss of vitreous collagen strands (C, closed arrow) into the supernatant. h, Representative TEM photomicrographs of supernatant from formalin–EDC-fixed vitreous tissue after incubation at 37ºC and heavy metal staining show few collagen strands (C, closed arrow), but no EVs in the supernatant. i, Representative images of a specificity control, tris-buffered saline (TBS) alone, show no collagen fibers nor EVs in the supernatant, but do show non-specific punctate staining of electron dense foci (NS, open arrow) measuring less than 20 nm. j, Graph demonstrating mean ± standard deviation concentration of total EVs lost to supernatant (wash buffer) collected from formalin-fixed bovine vitreous tissue after incubation at 37 ºC, 23 ºC or 4 ºC for 16 h. EV loss to the wash buffer was quantified using NTA and revealed substantial EV loss to wash buffers in formalin-fixed specimens incubated at room temperature (23 ºC) or elevated temperatures (37 ºC). At lower temperatures (4 ºC), the signal was below the NTA detection threshold. In tissues fixed with formalin–EDC, the amount of signal in the buffer contacting the fixed tissue was below the NTA detection threshold at 37 ºC. k–l, Graph demonstrating mean concentration (black line) ± standard deviation (red bars) according to EV size in the supernatant of formalin-fixed bovine vitreous after incubation at 37ºC (k) and 23ºC (l). Scale bars are (d) 200 nm, (e) 75 nm, (f) 200 nm, (h–i) 200 nm.