2.3.2. Scanning Electron Microscopy (SEM)-Alcian Blue Staining

HIV-1 Gag-eGFP VLP visualization by SEM was performed by staining VLP-containing supernatants with Alcian Blue solution, adapted from Gállego [17]. The protocol used is shown in Figure 2A. Briefly, Alcian Blue solution 1% w:v in 3% v:v acetic acid (Sigma Aldrich, St Louis, MO, USA) was diluted in ultrapure water achieving a final concentration of 1 μg/mL. Carbon-coated copper or Holey carbon 200 mesh grids were placed in this solution for 5 min, then the excess of Alcian Blue solution was removed by washing the grid in ultrapure water. Grids were dried with filter paper. Thereafter, 8 μL of the sample were placed on the grid and incubated at RT for 5 min. Excess sample was carefully drained off the grid with the aid of filter paper. Negative staining was applied to the samples when required, using the protocol described in Section 2.3.1. SEM micrographs were performed in a FE-SEM Merlin scanning electron microscope (Zeiss, Jena, Germany). Micrographs were taken with the

lens mode and secondary electron detector, with an electron high tension (EHT) comprised between 1–2 eV and 2.9–7.5 mm of working distance using a protocol adapted from González-Domínguez et al. [31].
