Congo Red Fluorescence Imaging of Amyloid Deposits

For Congo Red fluorescence imaging, the brains were fixed and washed as above then stained with 0.2 mg/ml Congo Red in PBS for 10 min. The Congo Red solution was freshly prepared and filtered through a 0.2 μm filter before use. After PBS washing, brains were observed using confocal microscopy. Congo Red fluorescence was detected as described (Wiesehan et al., 2003 (link)). LysoTracker red (Molecular Probes) staining was performed as previously described (Ling et al., 2009 (link)). For the CellMask plasma membrane staining, whole brains were first immunostained using anti-Aβ 4G8 (Covance) and Alexa Fluor-555-conjugated goat anti-mouse IgG (Invitrogen) secondary antibody, then washed in PBS, followed by a 10 min incubation in PBS containing 5 μg/ml CellMask™ Deep Red plasma membrane stain (Invitrogen). The brains were not permeabilized with detergent prior to the CellMask staining. After 5×PBS washing, brains were mounted and observed by confocal microscopy.