Formvar and carbon coated 200 mesh copper grids

Nanovesicles were isolated from the conditioned cultured medium of mouse calf muscle fibers (about 240 fibers), as described above. The 100,000 × g pellet was suspended in 2% paraformaldehyde in PBS and 5-μl aliquots deposited for 20 minutes on formvar- and carbon- coated 200-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA). Samples on grids were then fixed with 1% glutaraldehyde for 5 minutes, and negatively stained with uranyl acetate-oxalate solution for 5 minutes followed by embedding in methyl cellulose as described⁵⁵ . Samples on grids were viewed using a Hitachi H7000 electron microscope (Tokyo, Japan) at 75 kV. The electron microscope was equipped with an AMT Advantage HS digital camera (Danvers, MA, USA) and micrographs were digitally recorded.

Ultrathin sections (90 nm) were prepared using an ultramicrotome (EM UC7, Leica Microsystems) then mounted on formvar- and carbon-coated 200 mesh copper grids (Electron Microscopy Sciences).