H 7650 transmission electron

The virus particle integrity of the recombinant virus rSRV9-eGn was examined by transmission electron microscopy (TEM). Briefly, BSR cells were infected with rSRV9-eGn at a MOI of 0.1 for 72 h. The supernatant was collected, and viruses were recovered from cultures by pelleting the cell debris at 1500× g for 10 min. A drop of the supernatant was placed onto a copper-coated grid (mesh size 200) at room temperature. The grid was then removed, and the excess liquid was drained off by blotting the edge of the grid with a piece of clean filter paper. The grid was floated on a drop of 2% phosphotungstic acid (PTA) for 2 min and air-dried for a few minutes after the excess PTA was removed as before. The grid was viewed using a HITACHI H-7650 transmission electron microscope.

The harvested testes were promptly washed with 0.1 mol/L phosphate-buffered saline (PBS), immersed in 2.5% (w/v) phosphate-buffered glutaraldehyde and 4% (w/v) phosphate-buffered paraformaldehyde for 2 h at 4°C. Then, testes were washed with 0.1 mol/L phosphate buffer for 30 min, postfixed in 1% (w/v) phosphate-buffered osmium tetroxide for 2 h at 4°C. Samples were then embedded, sectioned, and double stained with uranyl acetate and lead hydroxide and analyzed using an H-7650 transmission electron microscope at 80 kV (Hitachi, Japan).

For transmission electron microscopy, cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), followed by 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h. Samples were blocked with 0.5% aqueous uranyl acetate overnight and subjected to low-temperature dehydration and infiltration with a graded series of Epon/Araldite, which was followed by the embedment in 100% Epon/Araldite. Thin sections (60 nm) were cut and stained with Reynalds lead citrate, and analyzed with a HITACHI H-7650 transmission electron microscope.

Exosomes were isolated from the USC supernatant by differential centrifugation/ultracentrifugation protocols. Briefly, the obtained medium was centrifuged at 300 g for 15 minutes and 2000 g for 30 minutes to remove dead cells and cellular debris. After centrifugation at 10 000 g for 1 hour, the supernatant was further ultracentrifuged at 100 000 g for 2 hours. After the removal of supernatant, the pellet was resuspended in phosphate buffer saline (PBS), followed by another ultracentrifugation at 100 000 g for 2 hours. Finally, pelleted exosomes were resuspended in PBS. Exosome morphologies were detected using transmission electron microscopy (TEM). Briefly, fresh exosome samples were loaded onto a continuous carbon grid, and then fixed in 3% (w/v) glutaraldehyde and stained with 2% uranyl acetate. The samples were then examined with a Hitachi H‐7650 transmission electron microscope (Hitachi). The size and concentration of the exosomes were assessed using flow nanoanalyser instruments according to the manufacturer's instructions.38, 39 Exosomal markers CD9 (1:1000; Abcam), TSG101 (1:1000; Abcam), Alix (1:1000; Abcam) and negative marker Golgi membrane bound protein (GM130；1:500; Abcam) were detected using Western blot analysis.

Electron microscopy was performed on 80 nm sections of pancreatic tissue from mice. All the sections were fixed in 2.5% glutaraldehyde (Sigma, G5882, Gillingham, UK) in 0.1 M phosphate buffer. The pancreas tissues were dehydrated using incremental concentrations of ethanol solutions and pure acetone before being embedded in the Spurr embedding medium. Subsequently, ultrathin slices were obtained using an Ultramicrotome Leica EM UC7 (Leica, Wetzlar, Germany) and stained with lead citrate and uranyl acetate. The mucosal ultrastructure was observed using a Hitachi H-7650 transmission electron microscope (Hitachi–Science & Technology, Tokyo, Japan).