Immunostaining of Phosphorylated Tau in Brain Sections

The OCT-embedded brains were cryosectioned at a 30 μm thickness. Staining was performed according to the previously reported method with a slight modification [50 (link),51 (link)]. The sections were permeabilized and blocked with 5% normal goat serum in 1X Tris-buffered saline (TBS) + 0.1% Triton-X (#9002-93-1, Millopore Sigma, St. Louis, MO, USA) for 1 h at room temperature. The sections were then incubated with a primary antibody against pTau Thr181 (1:500, #12885, Cell Signaling Technologies, Danvers, MA, USA) overnight at 4 °C and washed thoroughly with 1X TBS 3 times for 5 min. The sections were incubated in anti-rabbit AlexaFluor 488 (1:1000, #A11034, Invitrogen, Waltham, MA, USA) secondary antibody for 1 h at room temperature before washing with the same procedure. The sections were then stained with AlexaFluor 647 conjugated anti-NeuN (1:500, #D4G40, Cell Signaling Technologies, Danvers, MA, USA) antibody overnight at 4 °C before washing and treating with Hoechst (1:10,000, #33342, Invitrogen, Waltham, MA, USA). The sections were washed for 5 min 3 times with 1X TBS, coverslipped with Prolong Glass Antifade mountant (#P36930, Invitrogen, Waltham, MA, USA) and dried overnight at room temperature before storing at 4 °C. The slides were imaged on an LSM 800 confocal microscope (Zeiss, Jena, Germany).