Transmission electron microscope

The colon tissue was isolated and cut into small pieces and fixed in electron microscope fixative for 4 h, washed with 0.1 M phosphate buffer (Biogot technology, China) three times, post-fixed in 1% osmium tetroxide for 2 h. It was washed with 0.1 M phosphate buffer three times, dehydrated in graded solutions of ethyl alcohol (50%, 70%, 80%, 90%, 95%, 100%), and finally embedded in epoxy resin. Ultrathin sections were cut at a thickness of 60-80 nm. Stained samples were observed under a transmission electron microscope (Nikon, Japan), and images were collected and analyzed.

Exosomes were isolated from human serum using an exosome separation reagent (Invitrogen, Carlsbad, USA). In order to remove impurities, 500 μL of serum was centrifuged at 2000 × g for 30 min. Then, 300-μL samples were mixed with 60 μL of separation reagent and incubated at 4°C for 30 min. After centrifugation at 10,000 × g for 10 min, the supernatant was discarded. Next, 150 μL of phosphate-buffered saline (PBS) was added to the suspended sediment and placed in storage at 4°C for later use. The exosomes were fixed with paraformaldehyde (Sigma-Aldrich), added to the copper mesh, and negatively stained with uranium dioxide-acetate (Invitrogen). The morphology was observed using a transmission electron microscope (Nikon, Tokyo, Japan).