At100

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).

Immunofluorescence assay was performed as previously described (53 (link), 54 (link)). For hTau visualization, mouse antitotal hTau antibody (HT7; 1:1000) was used. To visualize phosphorylated tau, mouse antiphosphorylated hTau antibodies (AT8, AT100, AT180 [Thermo Fisher Scientific], and Tau-1 [Millipore Sigma]) were used. PHF-1 is a kind gift of Dr Peter Davies at the Feinstein Institute for Medical Research in Manhasset, NY. Tau 12 (Millipore Sigma) was used to detect the N-terminal end of tau. Primary antibodies were incubated overnight at 4 °C and detected with secondary antibodies for 1 h on ice (FITC or tetramethylrhodamine labeled; Invitrogen). Coverslips were mounted on glass slides and imaged using epifluorescence (Olympus 1X71). Images were captured by spot CCD digital camera (Diagnostic Instruments).
To determine the number of tau-positive neurons (i.e., HT7 or AT8 antibody) in our cultures, we counted the number of cells stained by an antibody per 1000 cells in each treatment group. Cells are counted using a nuclear dye 4′,6-diamidino-2-phenylindole (Molecular Probes) as previously described (53 (link)).

We used commercially available antibodies for the following antigens: Aβ (1:500, clone 6E10, BioLegend), γH2Ax (1:1000, #2577), H2Ax (1:1000, #7631), Histone H3 (1:1000, #9717), Caspase3 (1:1000, #9662), cleaved Caspase-3 (1:1000, #9661), α-Tubulin (1:1000, #2144, Cell Signaling Technology, MA), NeuN (1:500, EPR12763, abcam, UK), MAP2 (1:1000, clone Ap20, BD Biosciences, NJ), AT8 (1:1000, MN1020), AT180 (1:1000, MN1040), AT100 (1:1000, MN1060), ZO-1 (1:500, 40-2200), Tau5 (1:1000, AHB0042, Thermo Fisher Scientific, MA), γH2Ax (1:1000, host mouse, clone JBW301,#05-636), Tau-1 (1:1000, clone PC1C6, MAB3420), Olig2 (1:500, AB9610), β-actin (1:10000, A5441), H3K9me3 (1:1000, 05-499), Tau oligomeric (T22, 1:1000,#ABN454, millipore, CA), LaminB (1:1000, M-20, sc-6217), β Tubulin (1:1000, sc-5274), GAPDH (1:5000, FL-335, sc-25778, santa cruz biotech.), GFAP (1:500, G 3893, Merck, DE), mouse tau (1:1000, 012-26963), and Iba1 (1:500, 013-27691, FUJIFILM Wako Chemical Coporation, JP); mouse, rabbit and goat IgG HRP-conjugated (Jackson ImmunoResearch, PA).

To validate that intranuclear immunolabelling was not caused by artifact or non-specific staining, IHC was performed following the above protocol and using antibodies with the same isotypes for AT8 (mouse IgG isotype control; ThermoFisher Scientific™), AT100 (mouse IgG1 kappa isotype control; ThermoFisher Scientific™), and A15091A antibodies (mouse IgG2b kappa isotype control; Biolegend™) as negative controls.