All reagents for Thioflavin-S staining were purchased from Sigma-Aldrich, Natick, MA, unless otherwise indicated. Sections were rinsed with PBS for 5 minutes and then incubated in 0.05% potassium permanganate solution for 20 minutes followed by two washes in PBS and de-staining in 0.2% potassium metabisulfite and 0.2% oxalic acid (until the brownish color from the potassium permanganate is removed, less than 1 minute). The potassium metabisulfite/oxalic acid was washed out using PBS and sections were incubated in freshly-prepared 0.02% Thioflavin-S solution (in 40% ethanol) for four minutes in the dark. The remaining steps took place in the dark. Following staining, sections were developed with 50% ethanol for 15 minutes and followed by three time PBS wash and one time deionized water wash. Slides were coverslipped in Fluoromount-G and edges were sealed with nail polish. Fluorescent Thioflavin-S signals were imaged using a laser scan confocal microscope (LSM710, Carl Zeiss) by researchers blind to experimental condition. For each tissue section, we imaged five different regions.
