Anti at180

Twenty micrograms of protein of each sample were run on SDS-polyacrylamide gels (TGX gels, Bio-Rad), and transferred to nitrocellulose membranes by using the Trans-Blot® Turbo^™ Transfer System (Bio-Rad). The membranes were blocked with 5% BSA in Tris-buffered saline (TBS) plus 0.1% Tween-20 for 1 h, and then incubated with primary antibodies at 4°C overnight. The primary antibodies used were: anti-AT8 (Thermo Fisher, MN1020), anti-AT100 (Thermo Fisher, MN1060), anti-AT180 (Thermo Fisher, MN1040) and total Tau (C-terminal, Dako A0024). After washes with PBST, sections were incubated with Alexa 488- and 594-conjugated secondary antibodies (Invitrogen). The membranes were imaged using the ChemiDoc^™ system (Bio-Rad), and quantified with FIJI software.

Coronal free-floating Sects. (40 μm thick) were pretreated with 3% H2O2/3% methanol in Tris-buffered saline (TBS) for 30 min to block endogenous peroxidase activity. After TBS wash, sections were incubated first in TBS with 0.1% Trition X-100 (TBST) for 15 min, and then in TBST with 2% bovine serum albumin (BSA, Sigma-Aldrich) for 30 min. Sections were incubated with 6E10 (1:1000; BioLegend, San Diego, CA, USA), anti-HT7 (1:500; Thermo Scientific), and anti-AT180 (1:500; Thermo Scientific), in TBS + 5% normal horse serum overnight at room temperature. Sections were then incubated with biotinylated anti-mouse, 1:500 in TBS + 2%BSA + 5% normal serum for 1 h at 20 °C, followed by Vector ABC Kit and DAB reagents (Vector Laboratories, Burlingame, CA, USA) to visualize staining.
For double fluorescent stain, sections were incubated with anti-PSD95 (1:250; Invitrogen) and anti-synaptophysin (1:700; Sigma) overnight at 4 °C. Next, sections were incubated in secondary goat anti-mouse alexa-fluor 555 for synaptophysin and goat anti-rabbit alexa-fluor 488 for PSD95 antibody (Invitrogen) for 1 h. Sections were then mounted and coverslipped with Fluoromount-G (Southern Biothech).

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.