Veleta

For negative staining, a drop of exosomes was incubated for 1 min on a copper grid covered with formvar film, stabilized by carbon. Then, grids were exposed for 5–10 s on a drop of 0.5% uranyl acetate or 2% phosphotungstic acid. The grids holding the adsorbed exosomes, or ultrathin sections, were examined on a transmission electron microscope (TEM) (JEM 1400 (Jeol, Tokyo, Japan) with a digital camera Veleta (EMSIS, Münster, Germany)). Exosome measurements were made directly on the camera screen using iTEM (EMSIS, Münster, Germany) software.

Peptide solutions of 150 μM were diluted
with 10 mM AA (pH 7.3–7.4) or 10 mM AA solution enriched with
either 150 mM NaCl or 1.15 μM heparin and sonicated for 3 min.
Peptide samples were incubated at 37 °C for 9 days without agitation
prior to TEM imaging. Peptide solutions were then spotted on freshly
glow-discharged carbon/Formvar-coated mesh grids. After blotting off
the excess liquid, the samples were contrasted by 2% uranylacetate
(Polysciences Inc., Cat No 21447-25) in water for 1 min, the excess
stain was blotted off, and grids were air-dried. Fibrillar structures
were imaged on an 80 kV Tecnai 12 (Thermo Fisher) TEM at 135k and
300k magnification using a 2k × 2k pixel CCD side-mounted camera
(Veleta, EMSIS GmbH).

Hearts from freshly killed 12 week-old mice were removed, cut into halves and fixed in 2% paraformaldehyde, 2% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2. The left ventricle was cut in smaller cubes and subsequently post-fixed in 1% osmiumtetroxide, 1.5% potassiumferrocyanide in 0.1M cacodylate buffer, pH 7.2. The samples were stepwise dehydrated in ethanol, including en bloc 0.5% uranyl acetate staining during 70% ethanol incubation. Blocks were embedded in epon then sectioned ultrathin at 70 nm. Sections were collected on copper grids and stained with lead. The samples were analysed on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, The Netherlands) and representative areas were imaged with a 2 K CCD camera (Veleta, EMSIS, Münster, Germany).

Morphology of the isolated extracellular vesicles (EVs) was assessed by transmission electron microscopy (TEM) as described previously [26 (link)]. Specific markers for exosomes, CD9 or CD63 were detected as described earlier [26 (link)] using 10 μL of vesicles mixed with 10 μL of 0.5% bovine serum albumin in PBS and 3 μL (100 μg/mL) of corresponding monoclonal antibodies (Abcam, Cambridge, UK). All grids were examined in JEM 1400 (Jeol, Tokyo, Japan) TEM supplied with digital camera Veleta (EM SIS, Muenster, Germany). The measurements were made directly on the camera screen using iTEM (EM SIS, Muenster, Germany) software, version 5.2.
The size and concentration of the isolated EVs were determined by NTA using the NanoSight^® LM10 (Malvern Instruments, Malvern, Worcestershire, UK) analyzer, equipped with a blue laser (45 mW at 488 nm) and a C11440-5B camera (Hamamatsu Photonics K.K., Shizuoka, Japan), at several dilutions according to the manufacturer’s instructions. Each sample was measured in triplicate, with a camera setting of 15, an acquisition time of 60 s, and a detection threshold setting of 5. At least 200 completed tracks were analyzed per video. NTA analytical software version 2.3 (Malvern Instruments, Malvern, Worcestershire, UK) was used for data analysis and capture.