Tecnai g2 spirit biotwin transmission

EV pellets were fixed in 4% paraformaldehyde and pre-embedded with agarose. 1-mm³ cell blocks were mounted onto specimen holders and snap frozen in liquid nitrogen. 80 to 90 nm frozen sections were picked up with a 1:1 mixture of 2.3 M sucrose and 2% methylcellulose (15 cP) and transferred onto Formvar and carbon-coated copper grids. Sectioned slices were blocked with using 1% cold water fish-skin gelatin and incubated with primary antibody (anti-hThy-1: 1:200; anti-CD63: 1:50, see antibody sources below, in 1% BSA/PBS) for 1 h. Coverslips were washed three times in PBS (for 15 min each), incubated with secondary antibody (anti-mouse IgG-12nm gold and anti-rabbit IgG-18nm gold), washed three times with PBS (for 15 min each time). Grids were viewed using a JEOL 1200EX II (JEOL, Peabody, MA) transmission electron microscope and photographed using a Gatan digital camera (Gatan, Pleasanton, CA), or viewed using a Tecnai G2 Spirit BioTWIN transmission electron microscope equipped with an Eagle 4 K HS digital camera (FEI, Hillsboro, OR).

Control and UGCG-KO 293T cells cultured in a 6-well plate were incubated with HRTV (MOI = 50) at 4°C for 30 minutes and then at 37°C for 2 hours. Cells were harvested by cell scraper and fixed with 2.5% glutaraldehyde and 2% PFA. The representative images were collected by FEI Tecnai G2 Spirit BioTWIN transmission electron microscope.