Tecnai biotwin 120 kv

The different strains of fibrils were thawed, filtered using 100 kD columns (Millipore), and reconstituted. Uranyl acetate was used for negative staining. Proteins were loaded onto the carbon-coated grids and imaged by a transmission election microscope (FEI Tecnai BioTwin 120 kV).

Samples were diluted 1:500 in sterile phosphate buffered saline (HyClone, 10126473, Utah, UT, USA) and 5 µL diluted sample added onto carbon-coated grids (made in-house). After washing in distilled H₂O, the surface was blocked with bovine serum albumin (Sigma, A2153, Missouri, MO, USA) to prevent nonspecific binding and the specimens then incubated with 1:200 syn211 (sc-12767) for 1 h. Samples were again washed in distilled H₂O, incubated with 1:20 10 nm gold-anti-mouse antibody (Ted Pella, 15751, California, CA, USA), washed, fixed first with glutaraldehyde (Ted Pella 18427) and then uranyl acetate (Agar Scientific R1260A, Essex, UK). The samples then were examined under a transmission electron microscope (TEM) (FEI Tecnai Biotwin 120 kV, Minnesota, MN, USA).

For in vitro EM, lt-NES cell cultures (n = 8 wells) were fixed with 2% formaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Samples were frozen and cut into ultrathin sections with a diamond knife. Ultrathin sections were examined and photographed using a transmission electron microscope FEI Tecnai Biotwin 120 kv.
For in vivo iEM, 6 months after transplantation, three rats were deeply anesthetized with pentobarbital and transcardially perfused with 0.1 M PBS followed by ice-cold 2% formaldehyde, containing 0.2% glutaraldehyde, in 0.1 M PBS (pH 7.4). Brains were removed, post-fixed for 1 h and washed in 0.1 M PBS. For ex vivo iEM the same fixation was performed for 30 min. Both rat and human tissue were processed as described in supplemental experimental procedures.