Supplementary Figure 6: RNase treatment of formalin–EDC-fixed bovine vitreous tissue stained with propidium iodide (PI) show reduced extracellular nucleic acid signal.

a, Low-power wide-field fluorescent photomicrographs of whole mount bovine vitreous specimens crosslinked with formalin–EDC and stained with PI (a, top panel, red) show signal in the extracellular environment of vitreous tissue (denoted with closed arrowhead, inset); nuclei are labeled (a, middle panel, Hoechst, blue), and merged images are shown (bottom panel). Vitreous cell nuclei stain positive with PI (red) and Hoechst (blue); co-localized signals are shown (bottom panel, inset, green). Cells are denoted with an open arrow (a, middle and bottom panels, inset), and foci of extracellular PI signal are marked with a closed arrowhead (top and bottom panels, inset). Nuclei were stained, and no extracellular DNA signal is observed (bottom panel). b, Representative photomicrographs of whole mount bovine vitreous fixed with formalin–EDC, treated with RNase A, and imaged using the same exposure and microscopy settings as (a). Samples were stained with PI (top panel, red), Hoechst (middle panel, blue), and merged images are shown (bottom panel). RNase A treated samples show no evidence of extracellular RNAs as demonstrated by the lack of signal between the cell bodies (top and bottom panel) and show no signal between two cell nuclei (middle and bottom panels, open arrows Hoechst, blue). The PI signal for cytoplasmic RNA in RNase A treated samples (b, top and bottom panels) appear smaller than pre-RNase treated samples (a, top and bottom panels), presumably due to formalin–EDC retaining more cytoplasmic RNA. c, Graphical representation of mean ± standard deviation foci of extracellular signal for formalin–EDC fixed tissues stained with PI pre-RNase treatment and after RNase treatment shows significantly fewer extracellular vesicles after RNase treatment (n = 3 tissues, P = 0.0006 by Student’s two-tailed, unpaired t-test; t value 5.1, df 9). Scale bars are (a,b) 50 µm and (a inset, b inset) 20 µm.