For transmission electron microscopy, freshly isolated exosome suspensions were fixed in 4% paraformaldehyde for 1 hour. Exosome suspensions from different samples (approximately 5 μl) were applied to copper mesh Formvar coated carbon stabilized grids, were allowed to adsorb to the grid for 4–5 minutes and then were wicked off with filter paper. For negative staining of exosomes, 1% Aqueous Uranyl Acetate (5 μl) was applied to the grid for 30 seconds, then wicked off with Whatman filter paper. Grids were allowed to thoroughly dry before viewing.
As for immunoelectron labelling with anti-CD63 and anti-CD9, exosome samples were fixed overnight in 4% paraformaldehyde diluted in 0.1M cacodylate buffer (pH 7.4). Fixed exosome preparations (20 μl) were applied to a carbon-Formvar coated 200 mesh nickel grids, and samples were allowed to stand for 30 minutes before wiping off excess using Whatman filter paper. Grids were then floated (sample side down) onto a 20 μl drop of 1M Ammonium Chloride for 30 minutes to quench aldehyde groups from the fixation step, followed by floating on drops of blocking buffer (0.4% BSA in PBS) for 2 hours. Grids were rinsed 3 times (5 minutes each) using 1xPBS and then were allowed to incubate with either blocking buffer only (negative control) or primary antibody (CD63) diluted with blocking buffer (1:100) for 1 hour. Rinsing of the grids using deionized water (3 times for 5 minutes each) and 1xPBS followed the incubation step. Grids were then floated on drops of 1.4 nm anti-rabbit nanogold (Nanoprobes, Inc.) diluted 1:1000 in blocking buffer for 1 hour. Enhancing of grids using HQ Silver (gold enhancement reagent, Nanoprobes, Inc.) was then performed for 1 minute, followed by rinsing in deionized ice-cold water. As a final step, negative staining in 2% aqueous Uranyl Acetate was performed, and samples were wicked dry and then allowed to air dry. TEM examination was performed using JEM 1230 transmission electron microscope (JEOL USA Inc., Peabody, MA) at 110 kV and imaged with an UltraScan 4000 CCD camera & First Light Digital Camera Controller (Gatan Inc., Pleasanton, CA). TEM sample preparation and imaging was performed at the Electron Microscopy and Histology Core Laboratory at Augusta University (www.augusta.edu/mcg/cba/emhisto/ ).
As for immunoelectron labelling with anti-CD63 and anti-CD9, exosome samples were fixed overnight in 4% paraformaldehyde diluted in 0.1M cacodylate buffer (pH 7.4). Fixed exosome preparations (20 μl) were applied to a carbon-Formvar coated 200 mesh nickel grids, and samples were allowed to stand for 30 minutes before wiping off excess using Whatman filter paper. Grids were then floated (sample side down) onto a 20 μl drop of 1M Ammonium Chloride for 30 minutes to quench aldehyde groups from the fixation step, followed by floating on drops of blocking buffer (0.4% BSA in PBS) for 2 hours. Grids were rinsed 3 times (5 minutes each) using 1xPBS and then were allowed to incubate with either blocking buffer only (negative control) or primary antibody (CD63) diluted with blocking buffer (1:100) for 1 hour. Rinsing of the grids using deionized water (3 times for 5 minutes each) and 1xPBS followed the incubation step. Grids were then floated on drops of 1.4 nm anti-rabbit nanogold (Nanoprobes, Inc.) diluted 1:1000 in blocking buffer for 1 hour. Enhancing of grids using HQ Silver (gold enhancement reagent, Nanoprobes, Inc.) was then performed for 1 minute, followed by rinsing in deionized ice-cold water. As a final step, negative staining in 2% aqueous Uranyl Acetate was performed, and samples were wicked dry and then allowed to air dry. TEM examination was performed using JEM 1230 transmission electron microscope (JEOL USA Inc., Peabody, MA) at 110 kV and imaged with an UltraScan 4000 CCD camera & First Light Digital Camera Controller (Gatan Inc., Pleasanton, CA). TEM sample preparation and imaging was performed at the Electron Microscopy and Histology Core Laboratory at Augusta University (
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