Formvar carbon coated grid

To conduct TEM for exosomes morphology, the fixed exosomes in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) were moved onto the carbon/Formvar-coated grid (Ted Pella, Inc., Tustin, CA, USA) at room temperature for 30 min. After washing in PBS, the grid was cultured with 1% glutaraldehyde (Sigma-Aldrich), followed by the observation under JEOL JEM-2000EXII microscope (Jeol Ltd, Tokyo, Japan). To perform NTA for size distribution, exosomes were diluted to 10⁶ to 10⁹ particles/ml, then analyzed by NanoSight instrument (Malvern Panalytical, Malvern, UK).

Freshly isolated exosomes from feline AD-MSCs and feline fibroblasts were re-suspended in cold distilled water. Exosome suspensions were loaded onto a formvar carbon-coated grid (Ted Pella Inc., Redding, CA, USA) and fixed in 2% paraformaldehyde for 10 min, followed by the removal of the solution and drying of the sample. Grids were observed using a bioTEM instrument (HT7700; Hitachi, Tokyo, Japan).

The morphology
of the lipid bilayer discs was examined using a JEM-2200FS transmission
electron microscope (JEOL) specially optimized for cryo-TEM at the
National Center for High Resolution Electron Microscopy (nCHREM) at
Lund University. It is equipped with a Schottky field-emission electron
source and operated at an acceleration voltage of 200 kV. An in-column
energy (omega) filter and a 25 eV slit were used. The images were
recorded via SerialEM software under low-dose conditions onto a bottom-mounted
TemCam-F416 camera (TVIPS).
Each sample was prepared using an
automatic plunge-freezer system (Leica EM GP) with the environmental
chamber operated at 20 °C and 90% relative humidity. A droplet
(4 μL) taken from a sample that had been incubated at 37 °C
was deposited on a lacey Formvar carbon-coated grid (Ted Pella) and
was blotted with filter paper to remove excess fluid. The grid was
then plunged into liquid ethane (around −184 °C) to ensure
the rapid vitrification of the sample in its native state. Prior to
the cryo-TEM measurements, the specimens were stored in liquid nitrogen
(−196 °C) before imaging under the microscope using a
cryotransfer tomography holder (Fischione Model 2550).

The H. pylori G27, Δhp1076 and ΔfliS strains were cultured in Brucella broth containing 0.4% cholesterol at 37°C under microaerophilic conditions for 2 days. The cells were then harvested and washed three times with phosphate−buffered saline (PBS). The cells were further resuspended in PBS to an OD600 of 10. Ten-microliter aliquots of the cell suspension were dotted onto a Formvar/Carbon coated grid (TED PELLA) and stained with 0.2% neutral phosphotungstic acid. Excess staining was removed using Whatman paper. Flagellation was examined under a transmission electron microscope (Hitachi H−7650) at 80 kV.

Viral particles were purified from 30 15-cm Petri dishes of iD98HR1 cells that had been treated with OHT for 3 days. The extracellular virions were collected from the culture supernatant. Cell debris was removed by centrifuging the supernatant at 6,000 g for 15 min. The intracellular viral particles were first released from cell pellets by three cycles of freeze and thaw. Viral particles in extracellular or intracellular fractions were concentrated by centrifugation at 130,000 g for 1 h on a 50% OptiPrep (Axis-Shield) cushion. The virions at the interface were collected, and the OptiPrep was adjusted to 25%. Subsequently, a gradient was generated by centrifugation at 350,000 g for 3 h with an NVT65 rotor (Beckman). Fractions of 1 ml were collected from the bottom of the tube. Proteins in each fraction were analyzed by immunoblotting with antibodies. Viral particles in the fractions were absorbed onto a formvar/carbon-coated grid (Ted Pella, Inc.), blotted dry, and negatively stained with 1% uranyl acetate for 15 min at room temperature. The morphology of the viral particles was examined, and images were obtained using a JEOL JEM-1200 transmission electron microscope.