Tecnai g2 spirit biotwin transmission electron microscope

The nsEM
imaging (Figure S2) was carried out on
the resuspended pellet of fixed viral particles from ultracentrifugation
by using the “Airfuge” method. This consists in the
ultracentrifugation of 85 μL of a dilution 1:5 in PBS of the
pelleted materials in Airfuge Beckman (Airfuge, Beckman Coulter Inc.
Life Sciences, Indianapolis, Indiana, U.S.A.) for 15 min at 21 psi
(82 000g).²⁹ The
grids were stained using 2% sodium phosphotungstate (NaPt), pH 6.8,
for 1.5 min, and observed with a Tecnai G2 Spirit BioTwin transmission
electron microscope (FEI, Hillsboro, OR, U.S.A.) operating at 85 kV.

N. fowleri trophozoites (2x10⁶) were treated with posaconazole at 0.2 μM, for 24 h and 48 h, washed with PBS and then fixed overnight at 4°C in modified Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate, pH 7.2).[72 ] 0.1% DMSO-treated controls were simultaneously processed. The samples were then post-fixed for 1 hour with 1% osmium tetroxide in 0.15 M sodium cacodylate, pH 7.4, dehydrated with an ascending series of ethyl alcohol and propylene oxide, and finally embedded in an Epon resin (Scipoxy 812, Energy Beam Sciences). Thin sections (50–60 nm) were cut using Leica UCT ultramicrotome, mounted on the Formvar and carbon-coated copper grids, and counterstained for 5 min with 2% uranyl acetate followed by Sato's lead stain for 1 min. Naegleria thin sections were examined using a Tecnai G2 Spirit BioTWIN transmission electron microscope (TEM) equipped with an Eagle 4k HS digital camera (FEI, Hilsboro, OR).

After incubation with 1000 ng NETs for 4 h, BMDMs (1 × 10⁶) were washed with cold PBS three times, and the cell pellets were fixed in 2.5% glutaraldehyde at 4 °C overnight. The cells were further postfixed with 1% OsO₄ for 1 h, dehydrated, embedded, and sectioned. The ultrathin slides were observed with a FEI Tecnai G2 Spirit Bio TWIN transmission electron microscope.

Isolated exosomes were resuspended and fixed with 2% paraformaldehyde. A drop of exosome suspensions (approximately10ul) was applied onto a formvar coated copper grid. Then, exosomes were negatively stained with 1% aqueous uranyl acetate for 2 min and wicked off with filter paper. Examination was operated using a FEI Tecnai G2 Spirit Bio TWIN transmission electron microscope (FEI. Hillsboro, USA).