Cm 12 electron microscope

Cultured cells were treated with EBSS for 1hr before fixed in 0.1M sodium cacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 hours, and then post-fixed with 1% osmium tetroxide and 1% potassium ferrocyanide for one hour at 40°C, and later block stained in 0.25% aqueous uranyl acetate, processed in a standard manner and embedded in EMbed 812 (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (60 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead citrate. Stained grids were examined under Philips CM-12 electron microscope and photographed with a Gatan (4k x 2.7k) digital camera.

Ultrathin-sections spanning the dorsal or ventral hippocampus were counterstained with uranyl acetate before viewing under the electron microscope (JEOL1200XL). Digitized images were captured using the AMT camera system, consisting of a 1.2 megapixel Hamamatsu CCD camera (Boston, MA) attached to JEOLXL1200 electron microscope, at magnifications ranging from 25,000x to 60,000x. Alternatively, images were digitally captured, using a Phillips CM12 electron microscope with Gatan 4 megapixel digital camera at a magnification of 25,000x.

For exosome morphology analysis, 5 μl isolated exosome were put on glow discharged carbon coated 400 mesh copper/rhodium grids and stained with 1% uranyl acetate aquous solution. For whole mount immune-electron microscopy, deposit 5μl of 2% paraformaldehyde fixed exosomes on glow discharged formvar-carbon coated copper grids, and let it adsorbed for 20min. After washing with PBS, the grids were incubated with 50mM glycine/PBS for 5min, blocked with 1% coldwater fish skin gelatin (Sigma) for 10min, then incubate with primary antibodies (anti-TSG101, Abcam) in blocking solution for 2 hours at room temperature. Following washing with PBS, gold conjugated secondary antibodies (15nm protein A- gold, Cell Microscopy Center, University Medical Center Utrecht, 35584 CX Utrecht, The Netherlands; 12nm colloidal gold-AffiniPure goat anti-rabbit IgG (H+L), Jackson ImmunoReasearch Laboratories, Inc., West Grove, PA) were applied in the blocking buffer for 1 hour. After washing with PBS, the grids were fixed in 1% glutaraldehyde in PBS for 5min, washed with water, contrasted and embedded in a mixture of 3% uranyl acetate and 2% methylcellulose in a ratio of 1 to 9. All stained grids were examined under Philips CM-12 electron microscope and photographed with a Gatan (4kx2.7k) digital camera (Gatan Inc., Pleasanton, CA)^{34 (link)}.