At180

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).

Brains were dissected into forebrain, hindbrain and cerebellum before processing by paraffin embedding as described [29 (link)]. Sections were analysed by immunohistochemistry using the phospho-specific antibody AT180 (Thermo Fisher) as described [30 (link)].

FFPE tissue was dewaxed in xylene, rehydrated in graded alcohols and then blocked with 10% H₂O₂ for 1 h at RT in the dark. Antigen retrieval was performed via microwave heating with citrate buffer pH-6 (tau5, AT180, anti-6X His tag), immersion in 80% formic acid for 1 h (4G8) or no treatment (AT8, MC1). Then sections were blocked with 10% normal goat serum in TBS-0.1% Triton X-100 for 1 h at RT, incubated with tau5 (1:200), anti-beta amyloid clone 4G8 (Biolegend, 1:2000), AT8 (Innogenetics, 1:500), AT180 (ThermoFisher Scientific, 1:1000), MC1 (kindly provided by Peter Davies, The Feinstein Institute for Medical Research, Manhasset, NY, 1:100), or anti-6X His tag (Abcam, 1:200) at 4 °C overnight, incubated with biotinylated goat anti-mouse/anti-rabbit IgG secondary antibody (Jackson Immunoresearch) for 1 h at RT, washed in TBS-0.1% Triton X-100, incubated with VectaStain ABC reagent (Vector Labs) for 1 h at RT, treated with 3,3′-Diaminobenzidine (DAB, Sigma) for 3 min, counterstained with hematoxylin, dehydrated in graded alcohols and xylene and mounted with DPX mounting reagent.
PK treatment was performed as an additional step after antigen retrieval by incubating slides in 50 µg/ml PK (Sigma) 10 mM Tris HCl pH 7.8, 100 mM NaCl, 0.1% NP-40 at 37 °C for the stated times (0 s, 10 s, 1 min and 2 min).

Hippocampal brain tissue was homogenized in 10 volumes of Tissue Protein Extraction Reagent (TPER®, Thermo Scientific, catalog # 78510) with phosphatase and protease inhibitor cocktails (Sigma Aldrich, P5726 and P8340, respectively), and then sonicated on ice. Soluble hippocampal lysates were centrifuged at 12,000×g for 30 min at 4° C and samples were prepared with LDS and RA (Thermo Fisher Scientific). Samples were resolved via NuPAGE 4–12% Bis-Tris Gels (Thermo Fisher Scientific) and immunoblotted overnight in transfer buffer.^{50 (link)} All blots were processed in parallel and derive from the same experiment. The dilutions of primary antibodies were as follows: AT8 (Thermo Fisher Scientific (MN1020)/PHF1 (a kind gift from Peter Davies, Albert Einstein College of Medicine) 1:10,000; AT180 (Thermo Fisher Scientific, MN1040) 1:5000; Tau5 (MAB-361)/Tau12 (MAB2241) obtained from Millipore and used at 1:20,000; SNAP-25 (a kind gift from the late Michael Wilson) 1:20,000; GAPDH (Millipore, AB2302) 1:20,000. Uncut blots can be found in Supplementary Fig. 4.

Following fixation with 4% PFA in 0.1 M phosphate buffer for 60 minutes, eyeballs were equilibrated in 30% sucrose overnight, and embedded in optimal cutting temperature compound (OCT). Cryosections (10 μm) were obtained, postfixed with 4% PFA for 10 minutes, rinsed with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 15 minutes at room temperature, and blocked with PowerBlock (BiogenX, San Ramon, CA, USA) for 1 hour. Next, sections were probed with the following primary antibodies: HSF1 (Enzo Life Sciences, Farmingdale, NY, USA), Hsp70 (Enzo Life Sciences), Grp78 (BD Biosciences), Chop (Santa Cruz Biotechnology, Dallas, TX, USA), p-Perk (Cell Signaling Technology, Beverly, MA, USA) and AT180 (Thermo Fisher Scientific). After washing with PBS, sections were incubated with appropriate AlexaFluor 488-conjugated secondary antibodies (Thermo Fisher Scientific), mounted with medium containing DAPI (Abcam, Cambridge, MA, USA) or nuclei were counterstained with propidium iodide (PI; Fisher Scientific International, Inc., Hampton, NH, USA). Images were taken with epifluorescence microscopy.