Digital imaging system

Cells were fixed in 1/2 strength Karnovsky's (2% paraformaldehyde/2.5% glutaraldehyde buffered with 0.2 M cacodylate) and postfixed in 2% OsO4 buffered in 0.2 M cacodylate buffer. After quick dehydration, cells were embedded in Epon 812 (Electron Microscopy Sciences), thin sectioned (70 nm), and stained with uranyl acetate for 2 hours and lead citrate for 5 minutes. The samples were imaged using a JOEL 1230 transmission electron microscope set to 80 kV, and the images were captured using a Gatan digital imaging system. Image analysis was performed using NIH Image J, for mitochondrial size and relative abundance. The latter was calculated by fraction of mitochondria area/cell area.

Exosomes were deposited on the carbon coated 200 mesh nickel grids (EMS) as above. After 2 min, excess fluid was removed and exosomes were fixed in Trump’s fixative for 10 min [21 (link)]. After washing with milliQ water (three times; 3 min each wash), grids were incubated with 50 mM acetate solution for 10 min to neutralize aldehyde groups. After washing with DPBS (three times; 3 min each wash), exosomes on grids were blocked in 5% donkey serum in DPBS for 30 min. Grids were incubated with anti-CD63 (1:250 dilution; 1 h) or DPBS in lieu of primary antibody (negative control) in a humid atmosphere. After washing with DPBS-Tween (DPBS containing 0.05% Tween 20) (six times; 3 min each wash), grids were first incubated for 30 min with AffiniPure Fab fragment and then with 18 nM gold conjugated AffiniPure donkey anti-mouse secondary antibody (1:20 dilution; 30 min). Grids were washed in DPBS-Tween as above (eight times; 3 min each wash) and negatively stained with 2% aqueous uranyl acetate. Grids were air dried and examined under transmission electron microscope (FEI Tecnai G2 Spirit BioTWIN) at an acceleration voltage of 80 kV. Digital images were captured with scale bar using a GATAN digital imaging system. All steps involving washings and incubations were carried out by inverting grids over a drop of water, buffer or diluted antibody solutions and performed at room temperature.