Supplementary Figure 9: Immunohistochemistry staining of extracellular vesicles (EVs)-specific protein TSG-101 in normal bovine vitreous.

a, Representative wide-field fluorescent photomicrographs of whole mount bovine vitreous specimens fixed with formalin, processed at cold temperatures, and placed in a custom chamber bathed in PBS solution demonstrate immunohistochemical stain for the EV-associated protein, TSG-101, in the extracellular space (left and middle, arrowhead, Alexa 488, green). The inset (all panels, top right) is a higher magnification image of the box in the middle (all panels). Nuclei are marked with Hoechst counterstain (left and right, blue, open arrow). Hundreds of punctate extracellular protein signals were observed (left and middle, Alexa 488, green). No evidence of extracellular DNA was observed in this normal tissue (right, Hoechst, blue). b, Representative photomicrographs from specificity controls for TSG-101 immunohistochemistry: whole mount normal bovine vitreous labeled with non-specific IgG antibody (secondary only antibody, Alexa488, green). The inset (all panels, top right) is a higher magnification image of the box in the middle (all panels). Signal was observed surrounding the nuclei (left and middle, Alexa 488, green). Images show no evidence of extracellular signal (left and middle). Nuclei are marked (left and right, Hoechst, blue). c, Proteomic data showed that GM130 protein was not present in the EV fraction. Representative wide-field fluorescent photomicrographs of whole mount bovine vitreous specimens fixed with formalin, processed at cold temperatures, and stained for the GM130 demonstrate no extracellular stain for GM130 (left and middle, Alexa 488, green). Nuclei are marked with Hoechst counterstain (left and right, blue). d, Graphical representation of mean ± standard error for TSG-101 signal in extracellular and intracellular spaces, *P = 0.008 based on a two-tailed, unpaired, Student’s t-test (n = 3 tissues, t value 11.1, df 2). e, Positive signal for TSG-101 is observed in the extracellular space of the formalin-fixed vitreous (left, Alexa 488, green). Nuclei are labeled with Hoechst (left, blue) and PI (right, red). There is no evidence of extracellular RNA in formalin-fixed samples (right, PI, red). Scale bars are (a, b) 40 µm, (a inset, b inset) 10 µm, (c) 20 µm, and (e) 10 µm.