Tissue Immunohistochemistry and staining

Human and mouse brain sections were deparaffinized, rehydrated through alcohols, and processed through antigen retrieval steps consisting of heat pretreatment in citrate buffer by either microwave or autoclave per antibody-specific protocols. Sections were treated for endogenous peroxidases with 3% hydrogen peroxide in PBS (pH 7.4), blocked in 5% non-fat milk in PBS, and incubated with primary antibodies overnight at 4 °C (see Table ​Table2).2). Biotinylated secondary antibody was applied for 45 min at room temperature. Finally, sections were incubated in an avidin–biotin complex with streptavidin-HRP (Vector’s Vectastain Elite ABC-HRP kit, Burlingame, CA) and the reaction product was visualized with 0.05% diaminobenzidine (DAB)/0.01% hydrogen peroxide in PBS. Negative controls consisted of full protocol except primary antibody. The presence of neurofibrillary tangles was assessed by Gallyas silver staining using standard methods. Digital images were obtained using a Leica DM6 microscope with a DFC 7000 digital camera (Leica Microsystems, Wetzlar, Germany) and imported into Adobe Photoshop (Adobe Inc, San Jose, CA).

Antibodies

pTau

Thr231

pTau

Ser422