Immunostaining Analysis of Tau and Microglial Markers

Immunostaining analysis was carried out as described^{26 (link),40 (link)}. The primary antibodies used were cis P-tau mAb (clone #25), hyperphosphorylated tau epitopes antibodies (AT8 and AT100) (Innogenetics), microglia-specific antibody (Iba1) (Wako), and neuonal nuclei specific antibody (NeuN) (Milipore). After treatment with 0.3% hydrogen peroxide, slides were briefly boiled in 10 mM sodium citrate, pH 6.0, for antigen enhancement. The sections were incubated with primary antibodies overnight at 4 °C. Then, biotin-conjugated secondary antibodies (Jackson ImmunoResearch) were used to enhance the signals. Manufacturer-supplied blocking buffer (Invitrogen) was used for each reaction. The sections were washed four times with TBS after each step. Labeled sections were visualized with a Zeiss confocal microscope. The gain of confocal laser was set at the level where there are no fluorescence signals including autofluorescence in sections without primary antibody but with secondary antibody (Fig. S10). Slides were imaged on a Leica SPE microscope (Leica Microsystems, Wetzlar, Germany) and VS120 slide scanner (Center Valley, PA). Immunostaining images and their co-localization were quantified using Fiji/ImageJ Coloc 2.