Abstract
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.
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Acknowledgements
We thank J. Moore for his technical support for this manuscript. This work was performed in part at the Weill Cornell Medicine Imaging Core (for Electron Microscopy), and Genomics Resources Core, Cornell NanoScale Facility, a member of the National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant NNCI-1542081). The Heed Foundation supported M.G. and J.T.G.P. The Rockefeller University Proteomics Resource Center acknowledges funding from the Leona M. and Harry B. Helmsley Charitable Trust for mass spectrometer instrumentation. T.T. was supported by an Extracellular RNA Communication grants U19CA179564 from the NIH. D.L. was supported by NCI R35 CA232093 Outstanding Investigator Award from the NIH. J.T.G.P. supported by Daedalus Fund at Weill Cornell Medicine, The Shulsky Foundation, Research to Prevent Blindness Foundation and Knights Templar Eye Foundation Inc.
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J.T.G.P., A.S.L., H.L.R. and S.M. conducted tissue fixation and recorded images with wide-field, confocal and MPM microscopy. J.T.G.P., S.O., H.L.R., A.R.F. and J.E.E. conducted electron microscopy and image analysis. J.T.G.P., G.B.A. and S.O. conducted transfection experiments. D.J.D. and M.P.G. contributed to human eye experiments. D.D.H. sectioned eye tissues. T.T. contributed to RNA experiments. J.T.G.P. and M.P.G. dissected the eyes for whole mount for imaging. H.M. and B.D.D. conducted proteomic analysis. C.M.K., S.T. and D.L. provided the mammary tumor model experiments and immunoblot data. R.P.R., M.P.G. and B.L. conducted statistical analysis. J.T.G.P. and M.P.G. wrote the manuscript.
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J.T.G.P., M.P.G. and D.J.D. have submitted a patent on the work. J.T.G.P. has licensed the technology from Cornell University and founded a biotechnology company, Aufbau Holdings Limited. Other authors have no conflicts of interest.
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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.
Supplementary Figure 2 Extracellular vesicles (EVs) are retained in bovine vitreous by crosslinking tissues with a non-reversible fixative.
a, Gross image of bovine vitreous placed on a vision testing card demonstrates the highly transparent, gel-like structure. b, Graph representing the mean ± standard deviation number of EVs per vitreous cell quantified from 3 images of 3 biological replicates show that formalin–EDC-fixed vitreous exhibit significantly more EVs than formalin-fixed vitreous. *P = 0.0007 by two-tailed, unpaired, Student’s t-test (n = 3 tissues and 3 images recorded per tissue, t value 4.3, df 8). c, Graphical representation of frequency distribution of bovine vitreous EV diameter as measured by multiphoton microscopy. Scale bars are (a) 1 cm.
Supplementary Figure 3 Formalin-EDC fixation of bovine vitreous retains extracellular vesicles (EVs) imaged with wide-field epi-fluorescent microscopy.
a,b, Low-power wide-field fluorescent photomicrographs of whole mount bovine vitreous specimens crosslinked with formalin–EDC (a) or formalin alone (b). a, Representative photomicrographs of bovine vitreous fixed with formalin–EDC and stained with CFSE to label protein (top and middle panel, white) and Hoechst to label nuclei (bottom panel, blue) show multiple round cellular foci (all panels, open arrowhead) with numerous extracellular protein signals (top and middle panels, closed arrowhead, CSFE, white) consistent with EVs. b, Representative photomicrographs of whole mount bovine vitreous fixed with formalin-only show nuclear stain (middle and bottom panels, Hoechst, blue) co-localizing with CFSE (top and middle panels, white), consistent with cellular DNA and nucleic acid (all panels, open arrowhead). There is no evidence of extracellular protein signal (top and middle panel, CSFE, white). Scale bars are (a,b) 100 µm.
Supplementary Figure 4 Protein staining with CFSE fluorescent dye is similar in formalin alone or formalin–EDC fixed human embryonic kidney (HEK293) cells.
a, Low-power wide-field fluorescent photomicrographs of cultured HEK293 cells crosslinked with formalin–EDC and stained with CFSE (a, top panel, green) shows positive signal in cells. The nuclei were labeled (a, middle panel, Hoechst, blue) and co-localized signals (a, lower panel) show that CSFE and Hoechst stain formalin–EDC fixed cells’ protein. b, Representative photomicrographs of HEK293 cultured cells fixed with 4% formalin, stained with CSFE and Hoechst, and imaged using the same wide-field microscopy settings as in Supplementary Fig. 3a. Formalin-fixed cells showed positive signal (b, top panel, CSFE, green) and Hoechst (b, middle panel, blue). Merged images show co-localization of both stains (b, lower panel). Scale bars are (a, b) 100 µm.
Supplementary Figure 5 Nucleic acid staining with propidium iodide (PI) is equivalent in formalin alone or formalin–EDC fixed cultured human embryonic kidney (HEK293) cells.
a, Low-power wide-field fluorescent photomicrographs of HEK293 cells crosslinked with formalin–EDC and stained with PI (a, left panel, red) shows positive signal in cells (n = 3 cell cultures). The nuclei were labeled (a, middle panel, Hoechst, blue) and co-localized signals (a, right panel, purple) show that PI and Hoechst penetrate the formalin–EDC fixed cell membranes. b, Representative photomicrographs of HEK293 cultured cells fixed with formalin (n = 3 cell cultures), stained with PI and Hoechst, and imaged using the same microscopy settings as in Supplementary Fig. 4a. Formalin-fixed cells showed signal for PI (b, left panel, red) and Hoechst (b, middle panel, blue). Merged images show co-localization of both stains (b, right panel). c,d, Low-power wide-field fluorescent photomicrographs of HEK293 cells crosslinked with formalin–EDC (c) or formalin alone (d), but without PI staining (negative control) show no evidence of signal nor auto-fluorescent cells (c,d, left panel). Cell nuclei were labeled (c,d, middle panel, Hoechst, blue) and merged images shown (c,d, right panel). Scale bars are (a–d) 100 µm.
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.
Supplementary Figure 7 Formalin–EDC fixation allows for detection of significantly more extracellular vesicles (EVs) near the cell surface from mouse mammary tumor tissue sections, as compared to normal mouse mammary tissue.
a–c, Representative transmission electron microscopy (TEM) photomicrographs of formalin–EDC-fixed normal mouse mammary tissue reveal few EV-shaped electron dense signals (closed arrowheads) in the extracellular space (ECS) near the cell surface in three biological replicates. d, Photograph of showing the gross anatomy of a formalin–EDC-fixed 4T1 mouse mammary carcinoma tumor dissected 3 weeks post transplantation into the mammary fat pad of a mouse (syngeneic graft) and prior to tissue sectioning and staining. e–g, Representative TEM photomicrographs of a formalin–EDC-fixed 4T1 mouse mammary carcinoma tumor cell line that was transplanted into the mammary fat pad of a mouse (syngeneic graft), dissected and processed the same as tissues in a–c, show substantially more EV-shaped electron dense signals located in the extracellular space (arrowheads, n = 3 tissues and 5 images captured per tissue), when compared with the healthy mouse mammary tissue controls. h, Graphical representation comparing the mean ± standard error of the number of EVs per area of extracellular matrix (ECM) counted by 2 different blinded observers across 3 biological replicates (n = 3, with 6 images captured per biological replicate at the same magnification) from cancer or healthy tissues. These data show significantly more EVs per ECM area in mammary tumor tissue than in normal mammary tissue (P < 0.001). Scale bars are (a) 1 µm (left) and 500 nm (right), (b-c) 1 µm, (d) 200 µm, (e) 100 µm (left) and 500 nm (right), and (f,g) 1 µm.
Supplementary Figure 8 Formalin–EDC fixation of cultured human embryonic kidney 293 (HEK293) cells permit imaging of extracellular vesicles (EVs) near the cell surface with transmission electron microscopy (TEM).
a, Western blot of cell lysates and isolated extracellular vesicles (EV iso) from HEK293 cells, probed for TSG101, CD81, CD9, or Syntenin 1 to verify for enrichment within EV preparation. Samples were tested for GM130 and cytochrome C as negative markers. Beta actin antibodies were used as a control. b, Nanoparticle tracking analysis (NTA) graphical representation of the size and concentration of extracellular vesicles (EVs) isolated from the culture media from HEK293 cells. The mean (black line) ± standard error (red bars) concentration of EVs according to EV diameter, based on NTA shows EVs are present in HEK293 media. c–e, Representative TEM photomicrographs of a formalin–EDC-fixed HEK293 tissue sections show EV-shaped electron dense signal in the extracellular space (closed arrowheads) near the cell surface in three biological replicates. (c) The photomicrographs from HEK293 replicate no. 1 show an EV near the cell body, and the inset (black box) depict the EV (arrowhead) at higher magnification (right). d, Representative TEM photographs from HEK293 replicate no. 2 show an outpouching of EV-like structure (bottom inset) connected to a cell membrane (middle, single arrowhead). The top inset shows an EV (black box) at the external to the cell (right, double arrowhead). e, A third biological replicate shows EV-like structures adjacent to the cell membrane (arrowheads). Scale bars are (c) 500 nm (left) and 100 nm (right), (d) 500 nm (left), 250 nm (middle) and 200 nm (right), and (e) 500 nm (left) and 200 nm (right).
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.
Supplementary Figure 10 Bovine vitreous is free of cells after low-speed centrifugation.
a, Representative low-power light photomicrographs of whole mount bovine vitreous after low-speed centrifugation followed by hematoxylin and eosin staining shows eosinophilic signal consistent with vitreous collagen (pink, arrow) without evidence of hematoxylin stained cellular nuclei. Scale bar is 50 µm.
Supplementary Figure 11 Human and bovine vitreous extracellular vesicles (EVs) transfer endogenous RNA into cultured cells.
a, Representative confocal photomicrograph images of a human retinal pigment epithelial cells (ARPE-19) after 24 h treatment with a bolus of bovine vitreous EVs that were pre-labeled with the nucleic acid stain acridine orange (AO). Images show uptake of labelled EV-RNA in ARPE-19 cells (left and right panels, AO, green). Nuclei are labeled (middle and right panels, Hoechst, purple), and a merged image (right panel) show transfection of ARPE-19 cells, with AO signal (green) in the cytoplasm. b, Graphical representation of mean ± standard deviation transfection efficiency (% of cells transfected) for ARPE-19 cells treated with bovine vitreous EVs for 3, 6, or 24 h (n = 3 cell cultures, *P = 0.00002 based on two-tailed, unpaired Student’s t-tests for ARPE-19 cells treated with labelled bovine EVs for 3 h versus control (t value 237.9, df 2), P = 0.0003 for ARPE-19 cells treated with labelled bovine EVs for 6 h versus control (t value 55.9, df 2), P = 0.00005 for ARPE-19 cells treated with labelled bovine EVs for 24 h versus control (t value 147.8, df 2)). c, Representative wide-field epi-fluorescent low-power photomicrographs of ARPE-19 cells treated with a 3 h bolus of EVs that were isolated from post-mortem human vitreous and pre-labeled with AO. Images show transfection of cells (left panel, AO, green). Nuclei were marked (right panel, Hoechst, blue). d, Graphical representation of of mean ± standard deviation transfection efficiency (% of cells transfected) for ARPE-19 cells treated with human vitreous EVs (n = 3 cell cultures, *P = 0.0001 for ARPE-19 cells treated with labelled human EVs for 3 h versus controls (t value 93.5, df 2) and P = 0.0002 for ARPE-19 cells treated with labelled human EVs for 24 h versus control (t value 64.3, df 2), based on a two-tailed, unpaired Student’s t-test). Scale bars are (a) 15 µm and (c) 100 µm.
Supplementary Figure 12 Delivery of recombinant bovine serum albumin (BSA) protein and recombinant green fluorescent protein (GFP) by bovine vitreous extracellular vesicles (EVs) to cultured human retinal pigment epithelial (ARPE-19) cells.
a, Representative photomicrograph of ARPE-19 cells treated with a bolus of bovine vitreous EVs that had been pre-loaded with 1 µg BSA conjugated to fluorescein by electroporation at 300 V show fluorescein staining (left, yellow) in the cytoplasm. Nuclei are labelled (middle, Hoechst stain, blue), and a merged image (right) shows substantial number of cells transfected. b, Representative photomicrograph of ARPE-19 cells treated with a bolus of bovine vitreous EVs that had been mixed with BSA-fluorescein without electroporation (0 V, control) show no fluorescein staining (left, green). Nuclei are labelled (right, Hoechst stain, blue). c, Graphical representation of mean ± standard deviation transfection efficiency (% of cells transfected) of ARPE-19 cells treated with vitreous EVs loaded with 3 µg, 1 µg, or 0.5 µg BSA-fluorescein by electroporation at 300 V, with EVs loaded with 0.5 µg BSA-fluorescein without electroporation (0 V, control), or with PBS alone without electroporation (0 V, control). *P < 0.0005 for each BSA-fluorescein dosage loaded at 300 V versus each control based on two-tailed, unpaired Student’s t-tests (n = 3 cell cultures for each, P =0.0001 for BSA-fluorescein 3 µg at 300 V versus BSA-fluorescein 0.5 µg at 0V (t value 88.3, df 2), P = 0.0001 for BSA-fluorescein 3 µg at 300 V versus PBS only at 0V (t value 88.3, df 2), P = 0.0002 for BSA-fluorescein 1 µg at 300 V versus BSA-fluorescein 0.5 µg at 0V (t value 70.0, df 2), P = 0.0002 for BSA-fluorescein 1 µg at 300 V versus PBS only at 0V (t value 70.0, df 2, P = 0.00003 for BSA-fluorescein 0.5 µg at 300 V versus BSA-fluorescein 0.5 µg at 0V (t value 199.0, df 2), P = 0.00003 for BSA-fluorescein 0.5 µg at 300 V versus PBS only at 0V(t value 199.0, df 2)). d, Representative photomicrograph of ARPE-19 cells after application of a bolus of bovine vitreous EVs that had been pre-loaded with 1 µg of recombinant GFP by electroporation at 300 V show positive GFP staining (left, green) in the cytoplasm. Nuclei are labeled (middle, Hoechst stain, blue), and a merged image (right) shows substantial number of cells transfected. e, Representative photomicrograph of ARPE-19 cells after application of a bolus of bovine vitreous EVs that had been mixed with GFP without electroporation (0 V, control) show no fluorescein staining (left, green). Nuclei are labelled (right, Hoechst stain, blue). f, Graphical representation of mean ± standard deviation transfection efficiency (% cells transfected) of ARPE-19 cells after application of EVs loaded with 1 µg or 0.5 µg GFP by electroporation at 300 V or 1 µg GFP without electroporation (0 V, control). *P < 0.005 for each GFP dosage loaded at 300 V versus each control at 0 V based on two-tailed, unpaired Student’s t-tests (n = 3 cell cultures for each, P = 0.0008 for GFP 1 µg at 300 V versus GFP 1 µg at 0V (t value 35.7, df 2), P = 0.0008 for GFP 1 µg at 300 V vs. PBS only at 0V (t value 35.7, df 2), P = 0.001 for GFP 0.5 µg at 300 V versus GFP 1 µg at 0V (t value 30.0, df 2), P = 0.001 for GFP 0.5 µg at 300 V versus PBS only at 0V (t value 30.0, df 2)). Scale bars (a, b, d, e) are 50 µm.
Supplementary Figure 13 Bovine vitreous extracellular vesicles (EVs) target the retina and deliver recombinant protein to mouse retina in vivo.
a, Representative confocal photomicrographs of mouse retina tissue sections after injection a dilute amount of bovine EVs (0.25 µg) loaded with recombinant bovine serum albumin (BSA) conjugated to fluorescein on day 3 post injection show signal in vitreous (green) that does not penetrate the inner limiting membrane. b, Representative confocal photomicrographs of mouse retina tissues section 3 weeks after injection of BSA-fluorescein-loaded EVs show signal in cells traversing the ganglion cell layer (GCL), the IPL (inner plexiform layer) and the OPL (outer plexiform layer, arrowhead, green). The inset box from (b) is shown in higher power in (c) demonstrating positive stain in a cluster of cells in the GCL and retinal nerve fiber layer. d, Representative confocal photomicrographs of mouse retina tissues 3 weeks after injection of PBS control show no fluorescein signal. (a–d) Nuclei were stained with Hoechst (blue, middle panels) and images merged in right panels. Abbreviations: outer nuclear layer (ONL), and inner nuclear layer (INL). Scale bars are (a) 30 µm, (b) 50 µm, (c) 25 µm and (d) 40 µm.
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Supplementary Figures 1–13 and Supplementary Tables 1–5
Supplementary Video
Nanoparticle tracking analysis (NTA) video demonstrates that extracellular vesicles (EVs) are lost from formalin-fixed bovine vitreous humor tissues, but undetected in formalin–EDC-fixed
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Gupta, M.P., Tandalam, S., Ostrager, S. et al. Non-reversible tissue fixation retains extracellular vesicles for in situ imaging. Nat Methods 16, 1269–1273 (2019). https://doi.org/10.1038/s41592-019-0623-4
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DOI: https://doi.org/10.1038/s41592-019-0623-4
