Tau 1

For all qualitative IF analysis of human brain tissue samples, freshly cut sections were prepared from late stage (Braak V–VI) AD and control patient tissues archived at the UNC Brain Bank. Paraffin embedded tissue blocks were sectioned at 7 µM thickness, adhered to slides, and deparaffinized in xylene. Slides were rinsed in ethanol and rehydrated (pure, 95%, 70%) in 1 X PBS. Sections were boiled for 10 min and incubated in hot Vectastain H-3300 citrate-based buffer for 30 min for antigen retrieval. Sections were then rinsed in 1X TBS, and permeabilized for 50 min in 2% TBS-Triton X-100 at RT. Blocking, primary, and secondary solutions matched the donor species of secondary antibodies used; sections were blocked in 1X TBS with 2% goat serum for 50 min. Sections were then incubated with primary antibody in 0.1% sodium azide solution for 48 h at RT, thoroughly rinsed in 1X TBS three times, and incubated in secondary antibody solution for 24 h at RT. DAPI staining was used to visualize nuclei. Primary antibodies and dilutions: Tau-1 (Millipore, MAB3420, 1:1000), GFAP 1:1000, (Dako, GA524). Secondary antibody dilutions: Alexa Fluor goat anti-mouse IgM μ-chain specific 488, 1:200 (Thermofisher, A-21042), Alexa Fluor goat anti-rabbit 568, 1:200 (Thermofisher, A-11011), Alexa Fluor goat anti-mouse IgG1 specific 647, 1:200 (Thermofisher, A-1240).

1,4-Dideoxy-1,4-imino-D-arabinitol (DAB), L-lactate, isofagomine, and internal control antibody anti-α-Tubulin mouse mAb (DM1A,1:5000) were purchased from Sigma (St. Louis, MO, U.S.A.). Primary antibodies of phosphorylated glycogen synthase kinase 3β (GSK3β) at Ser-9 (pS9, 1:1000) and phosphorylated Akt at Ser-473 (p-Akt, 1:1000) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Antibodies used to identify axons and dendrites, tau-1 (1:200), and antimicrotubule-associated protein-2 (MAP-2, 1:200) were purchased from Millipore (Billerica, MA, U.S.A.). Anti-GFAP antibody was purchased from Abcam (Cambridge, UK). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Japan. Neurobasal and B27 were purchased from Invitrogen (Grand Island, NY, U.S.A.)

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).