Ab64193

The cells grown on slides were fixed with 4% paraformaldehyde for 15 min and blocked with 1× phosphate buffer saline supplemented with 3 mg/mL bovine serum albumin, 100 mM glycine, and 0.25% Triton X-100 for 30 min. Subsequently, the slides were incubated with primary antibody rabbit anti-BDNF (1:500, ab108319), rabbit anti-light chain 3II (LC3II) (1:1000, ab48394), mouse anti-Tau (Tau-13, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-BRUCE (5 µg/mL, ab19609) at 4 °C, followed by culture with the combination of the fluorophore and Alexa Fluor^® 647 secondary antibody (1:200, ab150075) at room temperature for 1 h. All the antibodies used above were provided by Abcam except Tau. Then, the cell slides were added with 4′ 6-diamidino-2-phenylindole for nucleus staining, then immersed in distilled water, dried and observed under a fluorescence microscope (Zeiss, Thornwood, NY) or FV-1000 confocal microscope.
For morphological analysis, the cells were fixed with 4% paraformaldehyde for 15 min and incubated with tau antibody (1:200, ab64193, Abcam) for 1 h, followed by another incubation with fluorophore combined with Alexa Fluor^® 647 secondary antibody (1:200, ab150075) for 1 h. The cells were then observed under the fluorescence microscope, and the length of the main axon in each cell was measured by five independent experiments.

SDD-AGE was carried out as previously described13 (link) with minor modifications. Brain extract was thawed on ice. A 1.5% agarose gel was prepared by dissolving agarose in buffer G (20 mM Tris-Base, 200 mM glycine) and then adding 0.02% SDS. A total of 50 μg (for Tg4510 and Tg21221 brain extracts (Supplementary Fig. 8)) or 25 μg (for lambda phosphatase experiment (Fig. 7e)) of brain extract protein was incubated with 0.02% SDS sample buffer for a total of 7 min at R.T. just before loading. The SDD-AGE was run in Laemmli buffer (Buffer G with 0.1% SDS) at 30 V for 14 h or until the dye front reached the end of the gel. Protein was then transferred via capillary action to Immoblin P (Millipore) membrane at 4 °C for 16–24 h. Membranes were blocked in 5% non-fat dry milk/TBS-T for 1 h and then probed for total tau using rabbit polyclonal anti-tau antibody (#ab64193, Abcam, 1:4,000) overnight at 4 °C. Membranes were washed three times with TBS-T, probed with goat anti-rabbit IgG-HRP (#172-1019, Bio-Rad, 1:2,000) for 1.5 h at R.T., and washed three times with TBS-T. Membranes were developed using ECL kit (Western Lightning, PerkinElmer, USA) and detected on Hyperfilm ECL (GE healthcare, USA).

Sliced sections were air dried and incubated in frozen methanol (2 min) and in 4% paraformalaldehyde (4 min). After three washes in phosphate buffer saline (PBS) containing Tween 20 (PBS/T) (Sigma-Aldrich), the sections were incubated for 60 min in a blocking solution (normal goat serum, in PBS) and then overnight at 4 °C with the primary antibodies against neuropeptide Y (NPY) (mouse monoclonal anti-NPY antiserum (1:500), product code: sc-133080, Santa Cruz Biotechnology, Inc. Heidelberg Germany), brain-derived neurotrophic factor (BDNF) (rabbit polyclonal anti-BDNF antiserum (1:300), product code: sc-ANT-010, Alomone Labs, Jerusalem Israel), glial fibrillary acidic protein (GFAP) (mouse polyclonal anti-GFAP antiserum (1:200), product code: G3893, Sigma-Saint Louis, MO, USA) and tau protein (rabbit polyclonal anti-tau antiserum (1:1000), product code: Ab-64193, Abcam Israel). After three washes in PBS/T, sections were incubated for 2 h in DyLight-488 labeled goat anti-rabbit IgG (BNDF: 1/1250; tau: 1/1750) or in Dylight-594 goat anti-mouse IgG (NPY: 1/500; GFAP: 1/500; KPL, MD, USA) in PBS containing 2% normal goat or horse serum.

The remaining five mice were randomly selected from among group for perfusion sampling and frozen brain sections. The mouse anti-amyloid antibody (sc-28365, Santa Cruz Biotech), mouse anti-Phospho-p38 MAPK antibody (#9216, Cell Signaling Technology), rabbit anti-Tau (phospho, S262) antibody (ab64193, Abcam), rabbit anti-Synaptophysin antibody (ab32594, Abcam), sheep anti-Iba1 antibody (ab5076, Abcam), and rabbits anti-Tmem antibody (ab209064, Abcam) were used in the immunofluorescence, performed following the previously described protocols [12 (link)]. Six image areas were randomly selected according to the hippocampal region, Image Proplus 6.0, which was used to analyze the cumulative optical density as well as the positive cell number in the selected visual area.