Tecnai 12 tem

TEM imaging was performed to validate the presence of EVs in pellets EVs isolated from platelet-free plasma. The pellet of EVs was resuspended in PBS and diluted 1:10 in PBS for optimum imaging, applied freshly in discharged carbon Formvar 3 mm 200 Mesh Cu Grids (AgarScientific, Stansted, UK) for 2 min before being blotted with filter paper. Grids were stained with 2% uranyl acetate for 10 s, blotted and air dried. Grids were imaged in a FEI Tecnai 12 TEM at 12 kV (GAtan OneView CMOS camera, Pleasanton, CA, USA).

Exosome samples were adsorbed to glow-discharged carbon-coated 400-mesh copper grids for 3 minutes and then stained with 2% (weight/volume) uranyl acetate in water, rinsed briefly with water and air dried. The grids were visualized on a FEI Tecnai 12 TEM at 80 kV. Images were captured on a Gatan Orius CCD camera with Gatan Digital Micrograph software.

Exponentially growing cells were pelleted, loaded into 3 mm, 100 µm, or 200 µm carriers and high-pressure frozen using a Leica EM ICE. Samples were freeze substituted in a Leica AFS2 unit in 1% uranyl acetate +1% water in acetone. Samples were then maintained at − 45 °C for the remainder of the processing. Samples were washed with acetone and then ethanol for 15 min each, then infiltrated with HM20 resin (PolySciences) according to the following schedule: 25% resin in ethanol for 4 h, 50% resin in ethanol overnight, 75% resin in ethanol for 5 h, 100% resin for 6 h, 100% resin overnight, 100% resin for 8 h. Samples were then polymerized with UV light for 48 h, and gradually warmed to 0 °C after the first 24 h of polymerization. 90 nm ultranthin sections were acquired using a Diatome diamond knife with a Leica UC7 ultramicrotome and transferred to formvar-coated 100-mesh copper grids. Sections were post-stained for 10 min with 2% uranyl acetate, washed by passing over a series of warm water droplets, and then stained with Reynold’s lead citrate, washed, and dried. Grids were imaged in a FEI Tecnai 12 TEM operated at 120 kV using a Gatan OneView digital camera.

MDA-MB-435 cells were
plated onto Thermanox plastic coverslips (13 mm diameter; Thermo Scientific,
#174950) in six-well plates at a seeding density of 8 × 10⁵ cells and left to adhere overnight. Fresh medium was added
before incubation with 210 nM Match–AuNP or Match–AuNP–Tat.
After 24 h, the medium was removed, and cells were washed twice in
1,4-piperazinediethanesulfonic acid buffer (PIPES buffer; 0.1 M; pH
7.2; Sigma-Aldrich # P8203) for 5 min. The samples were prepared and
stained as previously described.^{29 (link)} After
staining, the sections were analyzed on a Tecnai 12 TEM and imaged
with a 16 megapixel Gatan OneView camera with CMOS sensors. Images
were obtained using a 1 s exposure time, and a pixel saturation of
5000–6000.

Microdissected cartilage from mice was fixed using 2.5% glutaraldehyde + 4% paraformaldehyde (stocks solutions both from Agar Scientific) in 0.1 M 1,4 Piperazine bis (2-ethanosulfonic acid) (Sigma-Aldrich) buffer at pH 7.2 at room temperature for 1–2 h, then at 4 °C until further processing. Samples were prepared as described previously [10] and imaged on an FEI company Tecnai 12 TEM with a Gatan US1000 camera. Thereafter, autophagosomes from images were counted from cells.