Multi-Modal Histological Staining Protocol

For immunohistochemistry staining, slides were thawed and treated with 0.03 % H₂O₂ in PBS to block endogenous peroxidase or fixed and permeabilized in acetone for immunofluorescence staining, blocked with casein (Vector Laboratories) in normal goat serum (Zymed), and then incubated with anti-CD45, phospho-Tau, ApoE, CD31, LRP-1, 6E10, GFAP, or NeuN primary antibodies. For immunohistochemistry, slides were then incubated with biotinylated goat anti-rat Ig (Jackson ImmunoResearch) and streptavidin-HRP (Zymed) and developed with an AEC (Red) substrate kit (Zymed), counter-stained with hematoxylin and mounted with Fluoromount-G. For immunofluorescence assay, slides were instead subjected to AF488, TexRed, or AF647 conjugated secondary antibody and coverslips were mounted with Vectastain containing DAPI (Vectorlabs). Standard or frozen histological tissue sections were formalin-fixed and processed for hematoxylin and eosin (H&E) or oil red-O staining and hematoxylin counterstaining, respectively, then examined by light microscopy. For the green fluorescent Thioflavine S (ThioS) staining of plaques, frozen sections were incubated with 1 % ThioS (Sigma-Aldrich) in distilled water for 5 min, differentiated in 70 % ethanol for 5 min, washed three times for 5 min each with distilled water and cover-slipped with Vectastain containing DAPI (Vectorlabs). Images were captured using a Zeiss Axio Imager M1 microscope. For further quantification of acquired images, Zen software (Carl Zeiss) was used to obtain intensities of signals and counting of positive signals. A detailed explanation on the method of quantification is described in each figure legend.