Anti at8

The animals were anesthetized with 3% sevoflurane for 5 min and perfused transcardially with PBS, followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for immunofluorescence of brain slices. The brain tissues of the mice were removed and stored at 4°C in paraformaldehyde. For immunostaining, 10 mm frozen slices of mouse brain hemispheres were used. The sections were incubated overnight at 4°C with anti‐ApoE (1:4000; Cat#178479, Calbiochem, Germany) and anti‐AT8 antibodies (1:1000; Cat#MN1020, Thermo Fisher Scientific, USA), followed by immunostaining with Alexa Fluor® 488 donkey anti‐goat IgG (1:500; Cat#A‐11055, Thermo Fisher Scientific, USA) and Alexa Fluor^® 594 goat anti‐mouse IgG (1:500; Cat#A‐11032, Thermo Fisher Scientific, USA) for 1 h at room temperature in dark. Finally, the slices were coated with 4′6‐diamidino‐2‐phenylindole, dihydrochloride (DAPI; Cat#104139, Abcam, UK) and incubated in a humidified dark room for 10 min before being observed in mounting fluid under a fluorescence microscope.

RIPA (radio immunoassay precipitation) extracts from mouse brain homogenates were used for Western blot analyses as previously described (Di Meco et al., 2014; Giannopoulos et al., 2013). Briefly, samples were electrophoresed on 10% Bis–Tris gels or 3%–8% Tris–acetate gel (Bio‐Rad, Richmond, CA, USA), transferred onto nitrocellulose membranes (Bio‐Rad), and then incubated overnight at 4°C with the appropriate primary antibodies; anti‐5LO [dilution: 1:200] (Santa Cruz, Dallas, TX, USA), anti‐HT7 [1:200] (Thermo, Waltham, MA, USA), anti‐AT8 [1:100] (Thermo), anti‐AT180 [1:200] (Thermo); anti‐AT270 [1:200] (Thermo), anti‐PHF1 (generous gift of Dr. Peter Davies); anti‐PHF13 [1:100 (Thermo)], anti‐SYP [1:300] (Santa Cruz), anti‐PSD95 [1:200] (Thermo), anti‐MAP2 [1:1,000] (Millipore), anti‐GSK3α/β [1:100] (Cell Signaling, Danvers, MA, USA), anti‐pGSK3α/β [1:100] (Cell Signaling), anti‐cdk5 [1:200] (Santa Cruz), anti‐p35/p25 [1:100] (Santa Cruz), anti‐GFAP (Santa Cruz), anti‐CD45 [1:100] (Thermo) and anti‐Beta actin [1:500] (Santa Cruz). After three washings with T‐TBS (pH7.4), membranes were incubated with IRDye 800CW‐labeled secondary antibodies (LI‐COR Bioscience, Lincoln, NE, USA) at room temperature for 1 hr. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). β‐Actin was always used as internal loading control.

Briefly, 20 fly heads for each genotype were homogenized in RIPA buffer, and lysates were loaded in each lane of 10% SDS gels and transferred to nitrocellulose membrane. Membranes were blocked in 5% BSA and incubated with primary antibodies at 4 °C overnight. After washing membranes with TBS-T, membranes were incubated with the appropriate secondary antibody. Using the ECL Western blotting detection reagent, membranes were developed and images were captured using FluorChem E image processor. Antibodies used were anti-Tau (1:1000, T46, Cat no. 13-6400, Invitrogen), anti-AT180 (1:1000, Cat no. MN1040, Invitrogen), anti-PHF-1 (1:1000, Cat no. MN1050, Invitrogen), anti-AT8 (1:1000, Cat no. MN1020, Invitrogen) and anti-β-actin (1:1000, Cat no. JLA20, DHSB). β-actin was used as a loading control. Signal intensity was quantified using ImageJ (NIH) software. Flies used were 30 days old after eclosion. For Western blot analysis of SH-SY5y cells, the following antibodies were used: anti-Tau (Cat no. ab64193, Abcam), anti-β-actin (Cat no. LF-PA0207, AB Frontier), anti-Myc (Cat no. C3956, Sigma), anti-HA (Cat no. H6908, Sigma), and anti-CHIP (Cat no. sc-133066, Santa Cruz Biotechnology).

We performed immunohistochemistry at the Penn Digital Neuropathology laboratory using AT8 antibody for phosphorylated tau (pre-)inclusions and NeuN for neurons using an identical protocol that was optimized for both cohorts. AT8 immunohistochemistry was performed using anti-AT8 (#MN1020, Invitrogen; dilution 1:1000) as primary antibody. NeuN immunohistochemistry was performed using anti-NeuN (#MAB377, Millipore; dilution 1:1000) and the signal was amplified using the Vector ABC Kit (#PK-6100). In a subset (3 legacy cases of the Erasmus cohort from 1993 to 1996, 2 cases of the Penn cohort processed externally; total N = 5), the quality of NeuN immunohistochemistry was suboptimal in all stained tissue sections in two repeated staining attempts, likely due to slightly different perimortem processing methods. In this subset, neuronal degeneration was evaluated based on haematoxylin staining rather than NeuN staining, as validated and described in detail below.

Immunostaining for brain slices was performed as described previously [7 (link)], in which the primary antibodies were also listed. New antibodies used in this study include: thioflavin S (MCE, HY-D0972, Monmouth Junction, NJ), anti-C12orf34 (aggregatin) (1:100, Abcam, ab122626, Boston, MA), anti-Aβ (1:100, Proteintech, 60342-1-Ig), anti-synaptophysin (1:100, Proteintech, 17785-1-AP), anti-AT8 (1:100, Invitrogen, MN1020, Carlsbad, CA), anti-ApoE (1:100, Abcam, ab20874), anti-pTau-S396 (1:100, Thermo Fisher Scientific, 44-752G), and anti-Secretogranin III (1:100, Proteintech, 10954-1-AP).