Ab1543

Tissue samples were lysed with RIPA containing the protease inhibitor PMSF and cocktail, on ice water and centrifuged at 12,000g for 10 min. The supernatant was collected, boiled, and the proteins were separated by SDS-PAGE and transferred to the nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder, the protein was labeled overnight with primary antibody at 4 °C. After 3 times wash with washing buffer, the second antibody was incubated at room temperature for 1 h followed by another 3 washes. The Odyssey system was used to observe the protein expression level. The primary antibodies used for Western blotting include: NR2A (1:1000; ab14596, Abcam, UK), NR2B (1:1000; SAB4300711; Millipore, USA), GluA1 (1:1000; 04-855, Millipore, USA), GluA2 (1:1000; MAB397, Millipore, USA), PSD95 (1:1000; 2507, Cell Signaling Technology, USA), Synapsin 1 (1:1000; AB1543, Millipore, USA), Synaptophysin (1:1000, s-5768,Sigma), Actin (1:1000; ab6276, Abcam), LaminB1 (1:1000; Abcam), NF-κB p65 (1:500; Cell Signaling Technology, USA).
The nuclear and cytoplasmic protein preparation kit (P1200, Pulilai) was used to separate the nuclear and cytoplasmic components according to the manufacturer’s procedures for subsequent experiments.

Free-floating hippocampal sections were washed 3×5 min in 0.01 M TBS, pre-incubated with 3% BSA, 0.1% Triton X-100 in 0.01 M TBS (blocking buffer) for 1 h and incubated with primary antibodies mouse anti-Aβ_1 - 16 (1 : 2000, 6e10, BioLegend), rabbit anti-BACE-1 (1 : 200, #5606, Cell Signaling), rat anti-CD68 (1 : 1000, #MCA1957GA, Bio-Rad), mouse anti-GFAP (1 : 10000, #G3893, Sigma), rat anti-lipocalin-2 (Lcn-2) (1 : 100, #70287, Abcam), and/or rabbit anti-synapsin-1 (1 : 1000, #AB1543, Millipore) at 4°C for 24 h. The next day, sections were rinsed 6×5 min in 0.01 M TBS and incubated with Alexa Fluor-conjugated secondary antibodies (AF488-conjugated donkey anti-rabbit, 1 : 500, #A21206; AF555-conjugated donkey anti-mouse, 1 : 500, #A32773 and/or AF488-conjugated donkey anti-rat, 1 : 500, #A21208, Invitrogen) for 2 h at room temperature in the dark. Then, sections were rinsed 3×5 min in 0.01 M TBS, mounted onto Menzel Superfrost glass microscope slides (Thermo Scientific) and coverslipped using Mowiol (Sigma) as mounting medium. Fluorescent images were obtained using the Leica DMI6000 B microscope (Leica Microsystems) at a 200xmagnification.

Neurons were fixed by 4% paraformaldehyde (Wako) for 1 h, permeabilized with 0.5% TritonX100 (Alfa Aesar, Lancashire, UK) for 6 min, and incubated in 1% bovine serum albumin (BSA, Sigma) for 1 h. The fixed cells were treated with mouse monoclonal anti-Tau-1 (axon marker, 1:1000, MAB3420, Millipore, Burlington, MA, USA), rabbit polyclonal anti-microtubule-associated protein 2 (MAP2, dendrite marker, 1:1000, AB1543, Millipore), guinea pig polyclonal anti-vesicular glutamate transporter 1 (vGlut-1, pre-synapse marker, 1:1000, AB5905, Millipore), or mouse monoclonal anti-PSD95 (post-synapse marker, 1:1000, ab2723, Abcam) primary antibody at 4 °C overnight. The neurons were then reacted with anti-mouse IgG-AlexaFluor488 (A11001, Invitrogen) or anti-rabbit IgG-AlexaFluor568 (A11011, Invitrogen) or anti-rabbit IgG-AlexaFluor647 (A-21245, Invitrogen) or anti-Guinea Pig IgG-AlexaFluor488 (A-11073, Invitrogen) for 1 h at room temperature.