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).
Corresponding Organization : University of Cambridge
Other organizations :
National Institutes of Health, National Institute of Neurological Disorders and Stroke, MRC Mitochondrial Biology Unit, NHS Blood and Transplant, Center for Genomic Science, Italian Institute of Technology, HUN-REN Institute of Experimental Medicine, University of Catania, Université de Bordeaux, Institut Polytechnique de Bordeaux, Centre National de la Recherche Scientifique, Semmelweis University, Fourmed Kft Gyógyház, Hutchison/MRC Research Centre, MRC Cancer Unit
Characteristics of the isolated EVs, such as their ultrastructure and morphology, as observed using TEM
control variables
Fixation of the EV pellets with 4% PFA-PBS for 10 minutes at room temperature, followed by overnight incubation at 4°C
Postfixation of the pellets in 1% OsO4 for 30 minutes
Dehydration of the samples in graded ethanol, including block staining with 1% uranyl acetate in 50% ethanol for 30 minutes
Embedding of the samples in Taab 812 resin and overnight polymerization at 60°C
Sectioning of the embedded samples
Analysis of the ultrathin sections using a Hitachi 7100 electron microscope equipped with a Veleta 2,000 × 2,000 MegaPixel side-mounted TEM CCD camera
controls
Positive control: None mentioned
Negative control: None mentioned
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