2.3.1. Transmission Electron Microscopy (TEM)-Negative Staining

Prior to negative staining, HIV-1 Gag-eGFP VLPs were concentrated by double sucrose cushion 25–45% (w:v) at 31.000 rpm and centrifuged for 2.5 h at 4 ◦C in a Beckman Optima L100XP centrifuge using a SW32 Ti rotor (Beckman Coulter, Brea, CA, USA). The 25–45% interphase was recovered and stored at 4 ◦C until analysis [41]. TEM micrographs were analyzed after air-dried negative staining. The protocol used is summarized in Figure 1A. Briefly, VLP samples were deposited onto carbon-coated copper or Holey carbon 200 mesh grids (Micro to Nano, Wateringweg, the Netherlands). Grids were glow discharged in a PELCO easiGlow glow discharge unit (PELCO, Fresno, CA, USA). Thereafter, 8 μL of sample were loaded onto the grid and incubated at RT for 1 min. Excess sample was carefully drained off the grid with the aid of filter paper. Samples were negatively stained with 8 μL of 2% w:v uranyl acetate by incubation at RT for 1 min. Excess stain was drained off as previously indicated and grids were dried at RT until analysis. TEM examinations were performed with a Jeol JEM-1400 (JEOL USA, Pleasanton, CA, USA) transmission electron microscope equipped with an ES1000W Erlangshen charge-coupled device camera (CCD) (Model No. 785; Gatan, Pleasanton, CA, USA).
