Phospho tau at8

Neuronal cells, hiPSC-derived organoids, and mouse brain slices mounted on glass slides, were fixed with 4% paraformaldehyde. Samples were blocked for 1 hour with 2.5% BSA before incubating with primary antibodies at 4°C overnight. Primary antibodies used for IF were phospho-tau-AT8 (#MN1020, ThermoFisher), anti-tau-1 (#MAB3420, Millipore Sigma), eEF2, eEF2K, β-tubulin (#2332, #3692S, #2146S, Cell Signaling), Nestin (#NB100–1604, Novus). Samples were washed with PBS containing 0.1% TritonX-100 (PBST) prior to incubation with AlexaFluor secondary antibodies for 1 hour at room temperature. Images were captured on Cytation 5.0 under identical capture settings. High resolution images (60X silicon) were captured using the Andor Spinning-Disk Confocal microscope. Image analyses were conducted using QuPath open source software.

PFC punches were collected from brain slices and homogenized in 1% SDS buffer. SDS electrophoresis and transferring were performed to detect target proteins by incubating overnight with the following primary antibodies: Phospho‐Tau (AT8) (1:500, Thermo Fischer, MN1020), Tau (Tau‐5) (:500, Thermo Fischer, AHB0042), or GAPDH (1:2000, Cell Signaling, 5174). After secondary antibodies (horseradish peroxidase‐conjugated) incubation, enhanced chemiluminescent (ECL) reaction was performed using ECL substrate (SuperSignal™ West Femto Maximum Sensitivity Substrate or SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Scientific). Luminescence was detected by Chemidoc XRS system (Bio‐Rad), and density of blots was quantified by ImageJ software (NIH).