Ps214

Sagittal parallel sections of paraformaldehyde (PFA)-fixed female mouse brains were stained with antibodies specific for Aβ₄₂ (6E10, BioLegend, San Diego, CA, USA; McSA1, MédiMabs, Montreal, QC, Canada; MOAB-2, MilliporeSigma, Billerica, MA, USA) to detect intraneuronal Aβ₄₂ deposition and amyloid plaques in the hippocampus and cortex of the mice. To stain for tangle pathology, we used HT7, AT8, AT100, AT180, and AT270 (Thermo Fisher Scientific, Waltham, MA, USA) and T22 (MilliporeSigma); anti-tau antibodies pT231, pS214, and pS404 (Abcam, Cambridge, MA, USA); and Tyr18 (MédiMabs). NeuN antibodies (clone ABN78, MilliporeSigma; clone 1B7, Abcam) were used to stain neurons. Prior to the staining, sections were treated with heat-mediated antigen retrieval for all the tau antibodies or incubation in 70% formic acid for all the Aβ antibodies. After staining, tissues were scanned using a NanoZoomer digital pathology system and analyzed with NDP.view software (both from Hamamatsu Photonics, Shizuoka, Japan).

Proteins were separated on 10 % (w/v) SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking, membranes were incubated overnight at 4 °C with primary antibodies directed against total tau (rabbit polyclonal, 1/10,000, DAKO; mouse monoclonal CP27, 1/10,000, gift from Peter Davies), GST (goat polyclonal, 1/2000 GE Healthcare), phosphorylated tau (mouse monoclonal PHF-1, 1/5000, and CP13, 1/400, both gifts from Peter Davies; rabbit polyclonal pS214, 1/500, Abcam; rabbit polyclonal pS262, 1/500, Abcam), dephosphorylated tau (mouse monoclonal Tau-1, 1/5000, Millipore), fyn (rabbit polyclonal, 1/1000, HPA023887, Sigma), β-actin (mouse monoclonal, AC15, 1/10,000, Sigma), and neuron-specific enolase (mouse monoclonal, BBS/NC/VI-H14, 1/10,000, DAKO). Blotted membranes were incubated with secondary antibodies (Alexa Fluor® 680 goat anti-mouse, 1/10,000, Life Technologies or IRDye™ 800 conjugated goat anti-rabbit, 1/10,000, Rockland Immunochemicals Inc.) and antigens were visualised and quantified using an Odyssey scanner (Li-Cor Biosciences).