G box f3 genesys

Exosomes were fixed in 2% glutaraldehyde in DNase/RNase-Free Distilled Water (for 10 min on 150 mesh formvar and carbon-coated copper grids (Società Italiana Chinici, Rome, Italy), and dried under a hood. The images were acquired with a transmission electron microscopy (TEM) using a Morgagni 268D electron microscope (Philips, Andover, MA, United States) operating at 80 kV and equipped with a Megaview II camera (Olympus corporation, Tokyo, Japan) for digital image acquisition.
To perform Western blot of ASCs-exosomes, the proteins were denatured, separated on 4-12% polyacrylamide gels, transferred onto a nitrocellulose membrane and probed with antibodies against Alix (1:50, Santa Cruz Biotechnology, Q-19:sc-49268), heat shock protein 70 (HSP70, 1:100 Santa Cruz Biotechnology, sc-1060), and tetraspanine CD81 (1:100 Santa Cruz Biotechnology, sc-9158). The appropriate HRP-conjugated secondary antibodies against primary antibody (all secondary antibodies from Dako Agilent) were used. ASCs lysates were used as positive control. The blots were then incubated with a chemiluminescent HRP substrate and detected with G:BOX F3 GeneSys (Syngene, United Kingdom).

Analysis of exosomes by immunoblotting was performed using standard protocols: Proteins were denatured, separated on 4–12% polyacrylamide gels, transferred onto a nitrocellulose membrane and probed with antibodies against heat shock protein 70 (HSP70, 1:100 Santa Cruz Biotechnology, sc-1060), and tetraspanins CD9 (1:100 MM2/57, Millipore CBL-162) and CD81 (1:100 Santa Cruz Biotechnology, sc-9158) followed by appropriate horseradish peroxidase (HRP) conjugated secondary antibodies against the primary antibody (all secondary antibodies were from Dako Agilent). ASC lysates were used as the positive control. The blots were then incubated with a chemiluminescent HRP substrate and detected with G:BOX F3 GeneSys (Syngene, UK).