Megapixel side mounted tem ccd camera

For TEM analysis, EVs were generated and isolated from NSCs following the protocol described above [10 (link)]. Following the last ultracentrifugation step, the supernatant was carefully removed, and the pellets were fixed with 4% PFA-PBS for 10 minutes at RT, and then kept overnight at 4°C. After removal of the fixative and a short rinse with PBS, the pellets were postfixed in 1% OsO₄ (Taab, Aldermaston, Berks, UK) for 30 minutes, rinsed with distilled water, dehydrated in graded ethanol, including block staining with 1% uranyl acetate in 50% ethanol for 30 minutes, and embedded in Taab 812 (Taab). Overnight polymerisation at 60°C was followed by sectioning, and the ultrathin sections were analysed using a Hitachi 7100 electron microscope (Hitachi, Chiyoda City, Tokyo, Japan) equipped with Veleta, a 2,000 × 2,000 MegaPixel side mounted TEM CCD camera (Olympus, Shinjuku City, Tokyo, Japan).

In order to characterize the morphology and size of the different EV fractions, EV pellets were fixed with 4% paraformaldehyde in PBS for at least 60 min at room temperature and analyzed by transmission electron microscopy (TEM). After washing with PBS, the preparations were postfixed in 1% osmium tetroxide (OsO₄, Taab, Aldermaston, Berks, UK). This was followed by rinsing with distilled water. The pellets were dehydrated in graded ethanol including block staining with 1% uranyl-acetate in 50% ethanol for 30 min, and were embedded in Taab 812 (Taab). An overnight polymerization of samples at 60 °C was followed by sectioning, and the ultrathin sections were analyzed using a Hitachi 7100 electron microscope (Hitachi Ltd., Japan) equipped by Veleta, a 2000 × 2000 MegaPixel side-mounted TEM CCD camera (Olympus).

Six samples (2 aseptic, 2 low-grade infection and 2 acute purulent) were collected and analyzed by TEM. The aim of the TEM analysis was to visualize the vesicles in all types of samples (sterile, low-grade infection, acute infection) and investigate their properties.
The EV-containing pellet was fixed with 4% paraformaldehyde in PBS (pH 7. 5) for 2 hours at room temperature, rinsed with PBS and postfixed in 1% OsO₄ for 15 min. After a short rinse with distilled water, the pellet was dehydrated in graded ethanol including block-staining with 1% uranyl acetate in 50% ethanol for 15 min, finally was embedded in Taab 812 (Aldermaston, T031 UK). Following overnight polymerization at 60°C (Taab 812), 60 nm ultrathin sections were cut by a Leica UCT ultramicrotome (Leica Microsystems, UK) and analyzed with a Hitachi7100 (Hitachi, Japan) electron microscope equipped by Veleta, a 2 k × 2 k MegaPixel side-mounted TEM CCD camera (Olympus). Contrast and brightness of electron micrographs were adjusted by Adobe Photoshop CS3 (Adobe Photoshop Incorporation, CA).