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.