First light digital camera controller

Mock-infected or MCMV-infected RPE cells were treated with typsin for 5 min at 37 °C and centrifuged at 200 ×g for 5 min. After the supernatant was removed, the cell pellets were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCac) buffer, pH 7.4, postfixed in 2% osmium tetroxide in NaCac, stained en bloc with 2% uranyl acetate, dehydrated in a graded ethanol series, and embedded in Epon-Araldite resin. Thin sections were made using a diamond knife on a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany), collected on copper grids, and stained with uranyl acetate and lead citrate. Cells were observed under a JEM 1230 transmission electron microscope (JEOL USA, Peabody, MA) at 110 kV and imaged with an UltraScan 4000 CCD camera and First Light Digital Camera Controller (Gatan, Pleasanton, CA). Autophagic vacuoles were counted in individual cells from multiple fields and nonserial sections. Autophagic vacuoles were quantified by counting the number of autophagic vacuoles per cell.

TEM imaging of the isolated EVs was performed at the Electron Microscopy and Histology Core at Augusta University in Augusta, Georgia as previously described (Helwa et al., 2017 (link); Shah et al., 2018 (link)). Freshly isolated EV suspensions were applied to copper mesh Formvar coated carbon stabilized grids, fixed in 4% paraformaldehyde for 1 h, and stained with 1% aqueous uranyl acetate. After air drying, TEM examination was performed using a JEM 1230 transmission electron microscope (JEOL USA Inc., Peabody, MA, USA) at 110 kV and imaged with an UltraScan 4000 CCD camera & First Light Digital Camera Controller (Gatan Inc., Pleasanton, CA, USA).

EM analyses were performed at the High Resolution Electron Microscopy Facility of Medical College of Georgia at Augusta University. Livers were in situ fixed by perfusion with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, postfixed with 2% mOsO₄ (also in sodium cacodylate buffer) for 1 h. After dehydration, tissues were embedded in epon-Araldite resin. 70-nM thin sections were cut on a MT-7000 ultramicrotome and collected on copper grids. Sections were stained with uranyl acetate and lead citrate and examined under a JEM-1200 EXII transmission electron microscope (JEOL USA Inc.). Images were captured with an UltraScan 4000 CCD camera and First Light Digital Camera Controller (Gatan Inc.).