, 2000). All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Dissociated hippocampal neurons were plated onto poly-D-lysine coated coverslips (Carolina Biological Supply, Burlington, NC) or dishes at 20,000-40,000 cells/cm2 or 70-100,000 cells/cm2, respectively. Most experiments were performed at 19 DIV. Recombinant full-length and truncated α-syn with and without

a C-terminal myc-tag, were purified as previously described (Giasson et al., 2001). α-syn pffs were generated by incubating purified α-syn (5 mg/mL in PBS) at 37°C with constant selleck chemical agitation for 5 days, followed by aliquoting and storage at −80°C. The presence of amyloid was confirmed using Thioflavin T fluorometry. Fibrils of α-syn synthetic NAC peptide (amino acids 61-95) (Biotechnology Resource Center, Yale University) were generated as described (Giasson et al., 2001). Pffs were diluted in PBS at 0.1 mg/mL, sonicated several times, and diluted in neuronal media. For a 24-well tray, 1 μg/mL of pffs were added, and 5 μg/mL of α-syn pffs were added to a 60 cm dish. For this website WGA experiments, α-syn pffs were incubated with 1 μg/mL or 5 μg/mL WGA or preincubated for 1 hr (h) with 0.1 M GlcNAC followed by incubation with media containing

α-syn pffs, GlcNAC, and WGA. Neurons were fixed with 4% paraformaldehyde/4%

sucrose in PBS followed by permeabilization with 0.1% Triton X-100. To determine whether the pathologic α-syn aggregates were detergent insoluble, neurons were fixed with 4% paraformaldehyde, 4% sucrose, and 1% Tx-100 (Luk et al., 2009). Neurons were incubated in primary antibodies (Table S1) followed by Alex fluor-conjugated secondary antibodies (Invitrogen; Carlsbad, CA). Two-stage immunofluorescence was performed as described previously (Guo and Lee, 2011) to distinguish out between extracellular and intracellular α-syn-hWT pffs using mABs LB509 and Syn204. Unless otherwise stated in the figure legends, all experiments were performed a minimum of 3–10 times. Primary hippocampal neurons were fixed 14 days after addition of pffs. Neurons for transmission EM were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and postfixed for 1 hr in 1% OsO4 and 1.5% potassium ferrocyanide in 0.05 M cacodylate buffer. For immuno-EM, neurons were fixed in periodate-lysine-paraformaldehyde and permeabilized with 0.05% saponin in PBS with 2% fish gelatin (PBS-FG) and 0.05% thimerosal followed by incubation in mAB 81A. Neurons were then incubated in biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) followed by Avidin-Biotin Complex Elite (Vector Laboratories, Burlingame, CA) and postfixed for 15 min in 1.5% glutaraldehyde in 0.1 M cacodylate buffer with 5% sucrose and developed in DAB.