*3.2. Scanning Electron Microscopy (SEM)-Alcian Blue Staining*

SEM analyses were directly performed on harvested VLP supernatants from transfected HEK 293 (Figure 2B,D,F) and BV infected Sf9 cells (Figure 2E,G). Initial analyses using standard sample preparation protocols resulted in the loss of a high portion of the nanoparticles prior to visualization [47]. Thus, the combination of SEM with Alcian Blue solution was investigated (Figure 2) and compared to uranyl acetate staining as the reference methodology. The comparison between negative staining, Alcian Blue grid pre-treatment followed by negative staining, and only Alcian Blue grid pre-treatment is shown in Figure 2B–E, respectively.

Samples stained with uranyl acetate presented an important background regardless the grid treatment or not with Alcian Blue solution (Figure 2B,C and Figure S1). Indeed, large and irregular salt stacks deposited on the sample grid were detected probably related to the interaction of uranium with phosphate salts and amino acids from cell culture media, with similar results as observed in TEM micrographs (Figure 1). A decrease in the load of background signal was achieved by Alcian Blue grid pre-treatment without negative staining (white arrows) and nanoparticles could be individually resolved as 3D sphere-like structures. Despite the improvement in nanoparticle resolution achieved by the Alcian Blue grid pre-treatment, VLPs and EVs could not be distinguished in these analyses. Thus, the calculation of particle size distribution (PSD) was performed considering all nanoparticles as a single population, resulting in 296 ± 88 nm (*n* = 94) for HEK 293 and 162 ± 60 nm (*n* = 57) for Sf9 supernatants. The presence of EVs in conditioned supernatants could also be observed by SEM-Alcian Blue staining (Supplementary Materials S1), as well as the typical rod-shaped capsids of BVs (black arrows) in infected Sf9 supernatants (Figure 2E).

**Figure 2.** Scanning electron microscopy analysis of HIV-1 Gag-eGFP VLPs produced in HEK 293 and Sf9 cell lines harvested at 72 hpt and 72 hpi, respectively. (**A**) Sample preparation protocol; (**B**–**E**) Comparison of different sample preparation methods: (**B**) negative staining (NS) (**C**), combining Alcian Blue solution and negative staining (AB + NS) on HEK 293 supernatants; (**D**) Supernatant from transfected HEK 293 cells and (**E**) supernatant from BV infected Sf9 cells treated with Alcian Blue (AB) for 1 min in a Holey carbon 200 mesh grid. (**F**–**G**) PSD analysis of HEK 293 and Sf9 supernatants, respectively. White arrows indicate the presence of nanoparticles in HEK 293 and Sf9 supernatants and black arrows indicate the presence of BV. Negative controls were analyzed using the same conditions as in VLP samples (Supplementary materials S1).
