Jem 2100 plus electron microscope

The surface characteristics and morphology of XH-SLNs were observed by using HRTEM (JEM2100Plus Electron Microscope, JEOL company, Peabody, MA, USA). A drop of SLNs was stained negatively charged using 1% aqueous solution of phosphotungstic acid. This was placed on a micropipette having 200 micron mesh-size pioloform-coated copper grid. The film was dried for 1 h and analyzed under TEM at 50–80 kV [32 (link)].

For Transmission electron microscopy (TEM), MEF cells were cultured on aclar foil and fixed with pre-warmed fixative solution (2% glutaraldehyde, 2.5% sucrose, 3 mM CaCl₂, 100 Mm HEPES, pH 7.3) at RT for 30 min and 4°C for another 30 min. Afterward, fixed cells were washed with 0.1 M sodium cacodylate buffer, incubated with 1% OsO₄, 1.25% Sucrose, 10 mg/ml Potassium ferrocyanide in 0.1 M Cacodylate buffer for 1 h on ice, and washed three times with 0.1 M Cacodylate buffer. Subsequently, cells were dehydrated using ascending ethanol series (50, 70, 90, 3 × 100) for 7 min each at 4°C. After that, cells were incubated with ascending EPON series at 4°C: EPON with ethanol (1 + 1) for 1 h, EPON with ethanol (3 + 1) for 2 h, EPON alone ON, 2 × 2 h with fresh EPON at RT and finally embedded for 48–72 h at 62°C. Ultrathin sections of 70 nm were cut with a diamond knife using an ultramicrotome (Leica, UC7) and stained with uranyl acetate for 15 min at 37°C and lead nitrate solution for 4 min. Electron micrographs were taken with a JEM-2100 Plus Electron Microscope (JEOL). Camera OneView 4K 16 bit (Gatan), and software DigitalMicrograph (Gatan).

Neurons cultured on 18 mm Ø coverslips were fixed with pre-warmed fixative solution (2% glutaraldehyde, 2.5% sucrose, 3 mM CaCl₂, 100 mM HEPES, pH 7.4) at RT for 30 min, followed by the post-fixation at 4 °C for 30 min. Afterwards, the cells were washed with 0.1 M sodium cacodylate buffer, incubated with 1% OsO₄, 1.25% sucrose, 10 mg/ml K₄[Fe(CN)₆]·3 H₂O in 0.1 M cacodylate buffer for 1 h on ice and washed three times with 0.1 M cacodylate buffer. Subsequently, the cells were dehydrated at 4 °C using ascending ethanol series (50, 70, 90, 100%, 7 min each), incubated with ascending EPON series (EPON with ethanol (1 + 1) for 1 h; EPON with ethanol (3 + 1) for 2 h; EPON alone ON; 2 × 2 h with fresh EPON at RT) and finally embedded for 48–72 h at 62 °C. Coverslips were removed with liquid nitrogen and heat, consecutively. Ultrathin sections of 70 nm were made using an ultramicrotome (Leica, UC7) and stained with uranyl acetate for 15 min at 37 °C and lead nitrate solution for 4 min. Electron micrographs were taken with a JEM-2100 Plus Electron Microscope (JEOL), Camera OneView 4 K 16 bit (Gatan) and software DigitalMicrograph (Gatan).

FBS-EVs, saponin-EVs, and EV-ASTs were characterized using cryo-transmission electron microscopy (cryo-TEM). First, a holey carbon-coated copper grid with 200 mesh (Quantifoil, Großlöbichau, Germany) was glow-discharged to make it hydrophilic, and 3 µL of each sample was dropped and vitrified using a Vitrobot (Thermo Fisher Scientific, Waltham, MA, USA) by plunging into liquid ethane. The samples were then stored in liquid nitrogen and transferred to a cryoholder, which was maintained at −180 °C. A JEM-2100 PLUS electron microscope (JEOL, Tokyo, Japan) coupled to a CMOS camera (Thorlabs, Netwon, NJ, USA) at 25 kV was used to obtain TEM images.

NUGC4 cells were subjected to the indicated treatment and then underwent centrifugation at 5000 rpm for 5 min following trypsinization. Afterward, the cells were fixed with 2.5% glutaraldehyde at 4 °C for a duration of 2 h. Subsequent to fixation, the cells were postfixed with 1% osmium tetroxide under the same temperature conditions for 1 h. The cells were then dehydrated using a series of alcohol and acetone solutions of increasing concentration. Following dehydration, the cells were embedded in SPI‐Pon 812 (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were obtained by employing a Leica EM UC7 Ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA) and stained with uranyl acetate and lead citrate. TEM images were captured utilizing a JEM‐2100Plus Electron Microscope (JEOL Ltd. Tokyo, Japan).

The morphologies of FPV-SLNs were evaluated utilizing transmission electron microscopy (JEM-2100 Plus Electron Microscope, JEOL, Tokyo, Japan) with an acceleration voltage of 200 kV. TEM samples were planned by drop-casting FPV-SLN suspensions on a carbon-coated copper grid and air-dried before observations.

TEM of isolated EVs was performed at the Center for Electron Microscopy & Nanoscale Technology at Duke University Medical Center. Isolated EVs were diluted 1:100 in distilled water and immediately adsorbed onto Formvar‐ and carbon‐coated grids (Electron Microscopy Sciences). They were negatively stained with 2% aqueous uranyl acetate and examined in a JEM‐2100Plus Electron Microscope at 120 kV (JEOL USA). Micrographs were recorded on a 43 MP NanoSprint camera (AMT Imaging).