Transgenic adult female mouse overexpressing alpha-synuclein [548 (f) transgenic from D-line] was anesthetized with nembutal, and perfused with 4% paraformaldehyde + 0.1% glutaraldehyde, followed by 1 hr. postfixation in 4% paraformaldehyde. Tissue was sectioned on a vibratome at a thickness of 80µm. Following rinses in phosphate buffer (PBS), sections were blocked (PBS with 3% NGS;1% fish gel, 0.1% Triton X-1000; 1% BSA) for 1 hour, followed by rinses in working buffer (1:10 blocking buffer: PBS). Primary antibody was diluted in working buffer (anti-alpha-SYN; Host = Rabbit used at 1:500), and tissue was covered with aluminum foil and incubated on a shaker in cold room overnight. Tissue was washed with working buffer and incubated in secondary antibody (goat anti-rabbit AlexaFluor 568, 1:50) covered with foil, on cold room shaker, 48 hrs. After rinses, 1:100 Hoescht 33342 was used to stain nuclei (30 min). After rinsing in PBS, sections were mounted on slides and coverslipped using Gelvatol. Sections were imaged using a BioRad RTS 2000MP Multiphoton, with a Nikon Plan Fluor 40X, NA 1.3 objective.