Transmission electron microscope

L02 cells were harvested, fixed with 2.5% glutaraldehyde, washed twice with PBS for 30 min, fixed with 2.5% osmium tetroxide, and stained with 4% aqueous uranyl acetate. The cells were dehydrated with a graded series of acetone, embedded in epoxy resin, and excised into semithin and ultrathin sections. Electron micrographs were performed in the Central Laboratory of Third Military Medical University using a transmission electron microscope (FEI, U.S.A.).

Cells were harvested and fixed in 4% glutaraldehyde in 0.1 M PB (pH 7.4) for 24 h, followed by 1% osmic acid for 2 h. After fixation, cells underwent osmosis by acetone and 812 resins. Then, samples were embedded with Epon 812 and kept in a thermostatic drying oven for 4 h at 36 °C, 6 h at 45 °C, and 12 h at 60 °C. Afterwards, embedding blocks were successively cut into semithin sections (1–2 μm) and ultrathin sections (50–100 nm). Then, samples were stained with uranyl acetate and lead citrate. Finally, images were captured with a transmission electron microscope (FEI, USA) at an accelerating voltage of 80–120 kV.

All the cell samples collected were fixed with 2.5% glutaraldehyde overnight followed by 1% osmium tetroxide for 1.5 h. The cell samples were then dehydrated using a graded series of ethanol, rinsed with acetone and permeated overnight with embedding buffer. Sections of 70 nm thicknesses were dual-stained with 2% uranyl acetate and lead citrate. Adenovirus particles were examined by transmission electron microscope (FEI, Hillsboro, OR, USA).

The preparation and identification of SARS-CoV-2 VLPs are described previously (Mi et al., 2021 (link)). In brief, the codon-optimized S, E, and M genes of SARS-CoV-2 (GenBank accession no. MN908947.3) were cloned into the pFastBac triple expression vector. Recombinant baculovirus was produced using the Bac-to-Bac system (ThermoFisher Scientific, USA). VLPs were then obtained by infecting ExpiSf9™ insect cells with the recombinant baculovirus and purified via density gradient centrifugation.
SARS-CoV-2 VLPs were adsorbed onto a 400-mesh carbon-coated film for 2 min, which was then stained with 1% phosphotungstic acid for 60 s. After staining, VLP morphology was visualized using FEI Talos F200C transmission electron microscope (FEI, Czech Republic). For immunoelectron microscopy, VLPs were captured on carbon-coated copper grids. The grids were incubated with rabbit anti-spike polyclonal antibody (1:50 dilution; SinoBiological, China) at room temperature for 1 h, followed by treatment with goat anti-rabbit immunoglobulin G (1:20 dilution) (whole)-gold conjugate (10 nm) (BOSTER, China). Finally, the negative staining of the grids was performed using 1% phosphotungstic acid. VLPs were observed on the transmission electron microscope (FEI, Czech Republic) at 200 kV and 100–200 kfold magnification.