Mab348

The hippocampal sections were isolated and perfused with PBS and 0.2% Triton X-100 and then fixed in 4% paraformaldehyde for 24 h. The left brain sections were rapidly frozen under -50°C, followed by transversely cutting the tissue samples into 5 μm slices. The slices were then incubated at 4°C with anti-APP (1 : 200, MAB348, Millipore, USA) and BACE1 (1 : 300, ab183612, Abcam, USA) antibodies. After permeabilization and blocking overnight, appropriate secondary antibodies (ab150113, ab150115, Abcam, USA) were used at a dilution of 1 : 200. After washing three times with PBS, the sections were incubated with DAPI (C0065, Solarbio, China) for 10 min, followed by live imaging. The hippocampal images were captured and obtained with a confocal laser microscope (FV1000, Olympus, Japan) at 1000x magnification.

15–24 month-old Eu and TcMAC21 mice (n = 6 each) were perfused with PBS, then a half brain was used to make lysate for western blot and ELISA, and the other half was fixed in 4% PFA. WesternBlot and ELISA: cortex and hippocampus were removed and lysed with RIPA buffer. Protein extracts were separated by 4–12% SDS-PAGE, transferred to PVDF membranes and then probed with APP antibody (Millipore, MAB348) and beta actin. For ELISA, the above lysate was spun at 16,000 g for 30 min at 4°C. Supernatant was analyzed to determine Aβ levels by human/rat β amyloid ELISA kit from Wako (Aβ40: Cat# 294–64701; Aβ42: Cat# 290–62601). Amyloid plaque staining: mouse brains were fixed by immersion in 4% PFA, embedded in paraffin, and sectioned at 5 µm. Sections were deparaffinized and protein antigenicity was unmasked, and then the endogenous peroxidase activity was inhibited with 1.5% hydrogen peroxide. Sections were incubated with mouse anti-β-amyloid (Covance, Cat# SIG-39320), biotinylated goat anti-mouse IgG, and Avidin/Biotin mixture and then developed in 3,3′-Diaminobenzidine (DAB).

The following commercially available antibodies were used: anti-pTau (AT8; pSer202, pThr205) and total Tau (Tau 5) from ThermoFisher (RRID:AB_223647 and 10980631), anti-synj1 (rabbit polyclonal Ab, Novus; RRID:AB_11047653), anti-β actin and tubulin (Santa Cruz; RRID:AB_476697 and 477498), anti-holoAPP (MAB348, Millipore; RRID:AB_94882), anti-GSK3β (rabbit monoclonal Ab, Cell Signaling; RRID:AB_490890), anti-pGSK3β (Ser9, inactive form of GSK3β, rabbit polyclonal Ab, Cell Signaling; RRID:AB_331405), anti-pGSK3β (Y216, active form of GSK3β, rabbit polyclonal Ab, Abcam; RRID:AB_1310290), anti-mouse and rabbit horse radish peroxidase, Texas-Red or Alexa⁴⁸⁸ conjugated anti-mouse IgG (ThermoFisher; RRID:AB_2556542, 2540618, 10374713, 10983944, 2535987 and 1090271), were purchased. The pAb369 antibody which recognizes the C-terminus of APP was used to detect mouse holo-APP and c-terminal fragments^{67 (link)}.

A total of 10 µg of total protein obtained from tissue lysates or 20 μL of eluate biotin-conjugated protein was separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes according to the standard Western blot procedure. After the transfer, the membranes were incubated in TBS-T buffer (Thermo Scientific, Schwerte, Germany) with 5% bovine serum albumin (Fraction V) (Sigma Aldrich, Darmstadt, Germany) at RT for 60 min. Then, incubation with primary antibodies for β-actin (Sigma Aldrich, Darmstadt, Germany, Cat# A5441, 1:10,000), Alzheimer precursor protein A4 (Millipore, Darmstadt, Germany, Cat# MAB348, 1:2000), glutamate transporter 1 (Millipore, Darmstadt, Germany, AB1783, 1:3000) and tau (Abcam, Cambridge, UK, Cat# ab32057, 1:2000) was performed. Incubation was carried out at RT for 2 h. Secondary horseradish peroxidase (HRP)-conjugated antibodies were incubated with membranes at RT for 1 h (Promega, Cat# W402B and Cat# W401B, both 1:5000; Millipore, Cat# AP108P, 1:10,000). Detection was performed using SuperSignal West Pico PLUS blotting substrate (Thermo Scientific, Schwerte, Germany) and the GeneGnome XRQ analysis system (SYNGENE, Cambridge, UK). The data were analyzed using GnomeSys 1.8.2.