Cm10 transmission electron microscope

For TEM, 5 µl of the exosome-enriched isolate was placed on a carbon-coated 400-mesh grid for 30 s. The grids were washed twice in water and then stained with 2% uranyl acetate for 30 s. Imaging was performed using a CM10 transmission electron microscope (Philips Electron Optics, Eindhoven, The Netherlands) at 80 kV and images were collected using a Hamamatsu Orca HRL Camera (Advanced Microscopy Techniques, Woburn, MA, USA). For TEM analysis of samples isolated using the PEG, ultracentrifugation, and kit-based methods where the EVs samples were in PBS, samples were diluted 1:1 in fixation buffer, and then subjected to TEM analysis. Samples from the filtration method (above the filter) were diluted 1:1 in fixation buffer for the TEM analysis. A fixation buffer (e.g. 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4) was used for TEM analysis.

Transmission electron microscopy (TEM) images were made as described by Vallino et al. (54 (link)). Briefly, a phage suspension was adsorbed onto carbon and copper-palladium grids coated with Formvar for 3 min. Next, the grids were rinsed with water and negatively stained with 0.5% aqueous uranyl acetate. Samples were visualized using a CM10 transmission electron microscope (Philips, Eindhoven, The Netherlands) at a voltage of 80 kV.

Screening of gill samples with standard histology was performed on samples fixed in 10% neutral buffered formalin, followed by dehydration in an ascending alcohol series ending in xylol and afterwards embedded in paraffin. Paraffin blocks were cut in 2–3 μm thin sections, mounted on glass slides and stained using a routine protocol for haematoxylin and eosin (HE) staining.
For electron microscopy, formalin-fixed gill tissues were post-fixed in a mixed solution of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.5 at 4°C overnight. Samples were prepared for TEM by embedding into epoxy resin according to standard procedures (Seth-Smith et al., 2016 (link)). Epoxy resin blocks were screened for epitheliocystis lesions using semithin sections (1 μm) which were stained with toluidine blue (Sigma-Aldrich). Ultrathin sections (80 nm) were mounted on copper grids (Merck Eurolab AG, Dietlikon, Switzerland), contrasted with uranyl acetate dihydrate (Sigma-Aldrich), and lead citrate (Merck Eurolab AG) and investigated using a Philips CM10 transmission electron microscope. Images were processed with Imaris 7.6.1 (Bitplane, Oxford Instruments) and assembled into panels for publication and annotated using Photoshop (Adobe).

BNS and BNS-DOX samples were in vitro characterized by determining their average diameter, polidispersity index, and zeta potential with dynamic light scattering (90 Plus particle sizer, Brookhaven Instruments Corporation, Holtsville, NY, USA). The samples were diluted with water (1:30 v/v) immediately prior the measurements, carried out at a fixed angle of 90° and at a temperature of 25 °C. Transmission electron microscopy (TEM) analysis was performed using a Philips CM 10 transmission electron microscope to observe the morphology of BNS-DOX. The BNS nanosuspensions were sprayed on Formvar coated copper grid and air-dried before observation.
A weighted amount of freeze-dried BNS-DOX was dispersed in 5 mL of water and sonicated for 15 min. The sample was then centrifuged (10 min, 15,000 rpm) and after the supernatant was analyzed by High Performance Liquid Cromatography (HPLC), to determine the DOX concentration in the BNS.
The loading capacity of BNS-DOX was calculated according to: Loading capacity (%) = [amount of DOX/weight of BNS-DOX] × 100. The encapsulation efficiency of BNS-DOX was determined using the formula: Encapsulation efficiency (%) = [amount of DOX loaded/total amount of DOX] × 100.
The physical stability of BNS-DOX stored at 4 °C was assessed up to 1 month, by checking the physicochemical parameters over time.

The morphology of EVs was investigated with transmission electron
microscopy (TEM). For the analysis, a drop of 7 μL of the sample
was deposited on carbon and Formvar-coated 400 mesh grids (Ted Pella,
Redding, CA) previously glow-discharged with an Edwards E12E vacuum
coating unit (Burgess Hill, U.K.). After 5 min of incubation to allow
the adsorption of EVs, the grids were rinsed several times with water
and negatively stained with aqueous 0.5% w/v uranyl acetate. The excess
solution was then removed with filter paper.
Observations and
photographs were obtained using a Philips CM 10 transmission electron
microscope (Eindhoven, The Netherlands), operating at 60 kV. Micrograph
films were developed and digitally acquired with a D800 Nikon camera
at high resolution. Images were trimmed and adjusted for brightness
and contrast, and the scale was set using ImageJ 1.53c version.