PMC:5590178 / 12567-14091 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/5590178","sourcedb":"PMC","sourceid":"5590178","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5590178","text":"TEM was performed in the Electron Microscopy Facility at Yonsei Biomedical Reseach Institute at Yonsei University College of Medicine. In brief, zebrafish embryos injected with expression plasmids encoding WT or p.Arg79Cys GFAP were fixed at 30 hpf in 0.1 M phosphate buffer (pH 7.4) with 2% glutaraldehyde (Merck, Darmstadt, Germany) and paraformaldehyde (Merck) for 12 h, washed in 0.1 M phosphate buffer, post-fixed with 1% OsO4 in 0.1 M phosphate buffer for 90 min, dehydrated with an ascending ethanol series (50%, 60%, 70%, 80%, 90%, 95% and 100%) for 10 min each, and infiltrated with propylene oxide for 10 min. Subsequently, specimens were embedded with a Poly/Bed 812 embedding kit (Polysciences, Warrington, PA), polymerized in an electron microscope oven (TD-700, DOSAKA, Kyoto, Japan) at 65 °C for 12 h, cut into 200 nm thick semi-thin sections using an EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany) with a diamond knife (DiATOME, Hatfield, PA), stained with toluidine blue and observed with a light microscope. The region of interest was then cut into 80 nm thick ultra-thin sections using the ultramicrotome, placed on copper grids, stained in 4% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA) for 20 min followed by lead citrate (Thermo Fisher Scientific Korea) for 10 min, and imaged with a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) equipped with a MegaView III CCD camera (Olympus Soft Imaging Solutions, Lakewood, CO) at the acceleration voltage of 80 kV.","tracks":[]}