H 7650 transmission

Scalp needle (No. 7) was inserted into the trachea from centripetal direction and fixed with silk thread. Before residual lung gas pump back, RPMI 1640 (Gibco) supplemented with 5 % FBS (Gibco) slowly pushed into the trachea by syringe and lung tissue was put in sterile petri dish. 5 ml of RPMI 1640 supplemented with 5 % FBS was added into lung tissue, and lung tissue was grinded using centrifugal tube to prepare single cell suspension. Single cell suspension was added into centrifugal tube using mesh and discard the supernatant at 1500 r/min for 15 min. RPMI 1640 supplemented with 20 % FBS was added into cell and bilayer lymphocyte separation fluid was added into cell. After centrifuging at 2000 r/min for 20 min, cells of the upper and lower cell separation fluid interfaces were collected and fixed with Electron microscope fixative at room temperature for 30 min at darkness. Then, cell was observed using a Hitachi H7650 transmission electron microscope (Tokyo, Japan).
Cristae density was calculated by using Image pro for area void of cristae as previously described [52 (link)]. Mitochondrial size was measured using tracing individual mitochondrion after calibration for distance (minimum of 40 mitochondria).

Purified SUDV VLPs were processed and examined by Western blotting and transmission electron microscopy. For Western blotting, aliquots containing 10 μg of total protein were diluted with reducing buffer and denatured by heating at 95 °C for 10 min. Proteins were separated in 12% acrylamide gels, before they were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany) under denaturing conditions. For protein detection, two polyclonal antisera were used: mouse anti-SUDV GP polyclonal antisera and mouse anti-SUDV VP40 polyclonal antisera were mixed at a dilution of 1:1500 as a primary antibody for blotting; a goat anti-mouse IgG HRP-conjugated antibody (Bioss antibodies, Beijing, China) was used at a dilution of 1:5000 as a secondary antibody.
Negative staining transmission electron microscopy (TEM) was used to analyze the shape and size of purified SUDV VLPs. In short, 30 μL of sucrose gradient-purified SUDV VLPs were fixed for 15 min on carbon-coated formvar grids, grids were washed with 30 μL PBS and then treated with 1% phosphotungstate acid for 5 min. Grids were left to air dry and observed by using a HITACHI H-7650 transmission electron microscope.

High-titer phage lysates were prepared in LB medium based on the best MOIs of phages. The samples were then deposited on carbon-coated Formvar films and stained with 2% uranyl acetate. Microscopy was performed with a H7650 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan).

The lung tissue was minced into 1 mm³ cubes, fixed with 2.5% glutaraldehyde and 2% osmic acid, dehydrated and embedded in epoxy resin [21 (link)]. Ultrathin sections were collected onto 200-mesh copper grids, double stained with uranyl acetate and lead acetate, and then observed with a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan).

The morphology and characteristics of exosomes were verified by transmission electron microscopy using a H-7650 transmission electron microscope from Hitachi (Tokyo, Japan) as described [15 ].