Correlative Light and Electron Microscopy of U2OS Cells

U2OS cells stably expressing mGFP-Vpr grown on 35 mm gridded glass bottom dishes (MatTek Corporation) were subjected to light microscopy imaging. The resulting cells were fixed with Karnovsky’s fixative, then post-fixed, stained, and resin-embedded as described previously ^{81 (link)}, except that 2% OsO₄ and 1% uranyl acetate were used. In addition, a graded series of Polybed resin (EMS) with ethanol was utilized (2:1 ethanol to resin, 1:1 ethanol to resin, and 1:2 ethanol to resin each for 1 hour). Finally, the cells were incubated at room temperature for approximately 48 hours in resin without activator, followed by a 4-hour incubation in resin with activator BDMA at 32 °C. The cells had a final exchange with degassed resin and were allowed to polymerize for 24 hours at 65 °C. The resin was separated from the glass coverslip by heat shock, and any remaining glass particles were removed by hydrofluoric acid treatment and subsequent washing in ddH₂O. Regions of interest identified by light microscopy imaging were cut out with a jeweler’s saw and glued to resin blocks using super glue. Serial sections were cut en face on an ultramicrotome and collected on formvar-coated slot grids. Grids were post-stained with uranyl acetate and lead citrate according to standard protocols, then were carbon coated before being transferred to the TEM for tomography.