Leo em912 omega electron microscope

Exosomes were prepared from CSF as described above. The 100,000 × g pellet was fixed with 4% paraformaldehyde and adsorbed to glow-discharged Formvar-carbon-coated copper grids by floating the grid for 10 min on 5-µl droplets on Parafilm. The grids were negatively stained with 2% uranyl acetate containing 0.7 M oxalate, pH 7.0, and imaged with a LEO EM912 Omega electron microscope (Zeiss, Oberkochen). Digital micrographs were obtained with an on-axis 2048 _ 2048 CCD camera (Proscan, Scheuring).

For ultrastructural analysis of rat (postnatal day 4–p4) ED and ES, ES preparations including the ED were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 0.1 M sucrose for 30 min at 4 °C. Next, the specimen were washed three times with 0.125 M cacodylate buffer for 30 min. Afterwards, preparations were fixed with 1% osmium tetroxide (OsO₄) in 0.1 M cacodylate buffer for 1 h at room temperature, followed by dehydration in ethanol (50–100%). Next, the samples were transferred to propylen oxide (10 min propylen oxide (1:3), 30 min Epon propylen oxide (1:3), 30 min Epon propylen oxide (1:1), 60 min Epon propylen oxide (3:1) and finally 90 min in Epon (Epoxy Embedding Medium Kit; Sigma-Aldrich). The polymerization reaction was performed for 3 days at 55 °C. Ultrathin sections were counterstained with ethanolic uranyl acetate and lead citrate, and analyzed in a transmission electron microscope (LEO EM912 Omega electron microscope; Zeiss, Oberkochen, Germany). Images were obtained with a slow-scan CCD camera (PROSCAN, Germany; analySIS pro imaging software, version 3.2) and processed with Adobe Photoshop CS (Adobe Systems Software, Dublin, Ireland). For analysis of the TJ morphology 30 TJ of each part of the ED and ES were evaluated.