Talos l120c fei transmission electron microscope

A 200-mesh copper grid (MiTeGen, Ithaca, NY, USA) coated with a formvar/carbon film was subjected to the hydrophilic treatment. The iMSC-EVs suspension (4 μL) was placed on a grid and blotted for 1.5 min at 100% humidity and 4 °C. iMSC-EVs on the grid were visualized at 36,000× magnification using a Talos L120C FEI transmission electron microscope (Thermo Fisher Scientific) at 120 kV.

The left liver lobe, dorsal hippocampus, prefrontal cortex, and red gastrocnemius were selected as representative regions for the liver, brain, and skeletal muscle. Tissues were quickly dissected and cut into approximately 1 mm³ cubes in ice-cold PBS. Samples were fixed in 2.5% glutaraldhyde and 2% paraformaldehyde in 0.1M sodium phosphate buffer overnight, then rinsed in 0.1M sodium phosphate twice for 15 min. Tissues were post-fixed in 1% osmium tetroxide. After rinsing in distilled water, samples were dehydrated with a graded ethanol series from 50–100%. The samples were suspended in propylene oxide twice for 15 min and then pre-infiltrated overnight in 1:1 propylene oxide: resin (Dodecenyl Succinic Anhydride, Araldite 6005, Epon 812, Dibutyl Phthalate, Benzyldimethylamine) followed by infiltrating for 5 h in 100% resin. The samples were embedded in fresh resin and polymerized for 24 h at 70 °C. Approximately 100 nm sections were cut using a Leica EM UC6 ultramicrotome and collected on copper grids. The sections were stained with 4% aqueous uranyl acetate followed by 0.3% lead citrate in 0.1N sodium hydroxide and imaged with a FEI Talos L120C transmission electron microscope (Thermo Fisher, Waltham, MA, USA).

PFO mutants (0.025 mg/ml), CDCL (0.3 mg/ml), CDCL^S (0.05 or 0.2 mg/ml), or CDCL^S + CDCL (1:1 equimolar ratio, 0.05 or 0.2 mg/ml) in 20 mM HEPES (pH 7), 150 mM NaCl were applied to Teflon wells. The prereduced PFO mutants were treated with 2 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) reducing agent before incubation. A lipid mixture containing cholesterol:POPC (molar ratio 65:35 for PFO, molar ratio 55:45 for CDCLs) solubilized in chloroform (1 μl at 0.5 mg/ml) was then overlaid onto the wells and lipid monolayers were formed upon chloroform evaporation. Samples were incubated at 37°C for 60 min in a humidified chamber. Proteins assembled on the lipid layer were transferred onto Formvar/carbon-coated grids (mesh size 300) and stained with 1% uranyl acetate, following the method described by Rames et al. (43 (link)). All stained grids were imaged using an FEI Talos L120C transmission electron microscope (Thermo Fisher Scientific), with an acceleration voltage of 120 keV and fitted with a 4K × 4K Ceta CMOS camera at a magnification between 36,000× and 57,000× and a defocus of 0.05 to 2 μm.