Anti ht7

RIPA extracts from mouse brain homogenates were used for western Blot analyses as previously described^{14 (link),15 (link)}. Briefly, samples were electrophoresed on 10% Bis-Tris gels or 3–8% Tris-acetate gel (Bio-Rad, Richmond, CA), 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), anti-HT7 [1:200] (Thermo, Waltham, MA), anti-AT8 [1:100] (Thermo), anti-AT270 [1:200] (Thermo), anti-PHF13 [1:100 (Thermo)], anti-SYP [1:300] (Santa Cruz), anti-PSD95 [1:200] (Thermo), anti-GSK3α/β [1:100] (Cell Signaling, Danvers, MA), anti-pGSK3α/β [1:100] (Cell Signaling), anti-SAPK/JNK [1:100] (Cell Signaling), anti-pSAPKJNK [1:100] (Cell Signaling), anti-cdk5 1[:200] (Santa Cruz), anti-p35/p25 [1:100] (Santa Cruz), anti-PP2A [1:200] (Santa Cruz), anti-GFAP (Santa Cruz), anti-Iba1[1:100] (Thermo) and anti-Beta actin [1:500] (Santa Cruz). After three washings with T-TBS (pH 7.4), membranes were incubated with IRDye 800CW-labeled secondary antibodies (LI-COR Bioscience, Lincoln, NE) at room temperature for 1 h. Signals were developed with Odyssey Infrared Imaging Systems (LI-COR Bioscience, Lincoln, NE). β-Actin was always used as an internal loading control.

Anti-pT205 (SAB,11108-2), anti-pT231 (SAB,11110), anti-HT7 (Thermo, MN1000), anti-Tau5(Abcam, ab80579), β-actin (Abcam, ab6276), ChAT (Chemicon, AB144P), CaMKII (GeneTex, GTX127939), GABA (Sigma, A2052), DAPI (Beyotime, C1006), cholera toxin subunit B (CTB)555 (Molecular Probes, Eugene, USA)), Nissl staining solution (Beyotime, C0117), DAB-staining kit (ZLI-9031, ZSGB-BIO), atropine sulfate salt monohydrate (Sigma-Aldrich, A0257) and mecamylamine hydrochloride (Sigma-Aldrich, M9020).

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).