Rabbit anti app

After the behavioural tests, the mice were anaesthetized and transcardially perfused with saline. Then, the brains were removed, and the hemispheres were separated. Brain samples assigned for immunohistochemical staining were immersed in OCT at -80 °C before coronal sectioning on a Frozen slicer (Thermo Fisher Scientific, USA) (10 μm thickness). The sections were restored to room temperature, fixed with cold acetone for 5 min, and permeabilizing agent was used for 5 min. Then, the cells were rinsed three times at room temperature for 5 min each with 1x PBS (pH 7.2-7.4) and blocked with 10% bovine serum in 0.1% Triton X-100 for 30 min. The blocking solution covered all brain tissues and prevented the tissue from drying out. Then, the samples were incubated with primary antibodies overnight at 4 °C. The primary antibodies included rabbit anti-NeuN (1:500, Abcam), rabbit anti-GFAP (1:200, CST), rabbit anti-Iba-1 (1:100, Abcam), rabbit anti-CD68 (1:300, Abcam), rabbit anti-APP (1:100, Millipore), and mouse anti-β-amyloid specific for Aβ₄₂ (1:200, CST). The next morning, the sections were incubated for 1 h at room temperature with DyLight 488-/555-conjugated goat anti-rabbit/mouse IgG (1:200, Abcam) and stained with DAPI for 3 min. Fluorescence images were captured using an Olympus FV3000 confocal microscope.

Cerebral cortex and hippocampus extracts were loaded onto 10-16 % Tris/tricine SDS gels, and transferred to nitrocellulose membranes before overnight incubation with one of the following primary antibodies: rabbit anti-APP (1:1000; Millipore), rabbit anti-Aβ_1–42 (1:1000; Sigma-Aldrich, Saint Louis, MO, USA) and mouse anti-SAPPβ (1:1000; Sigma-Aldrich), mouse anti-CTFβ (1:1500; Millipore), mouse anti-BACE1 (1:2000; Millipore), rabbit anti-PS1 (1:1000; Sigma-Aldrich), rabbit anti-NEP (1:1000; Millipore), rabbit anti-IDE (1:1000; Abcam, Cambridge, MA, USA), mouse anti-GFAP (1:2000; Millipore), mouse anti-Iba-1 (1:500; Wako), mouse anti-SYP (1:1500; Millipore), rabbit anti-PSD-95 (1:1000; Abcam), mouse anti-BDNF (1:500; Sigma-Aldrich) or rabbit anti-β-tubulin (1:3000; Sigma-Aldrich). Horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories) were used, and bands were visualized using ECL plus detection system. β-tubulin was utilized as an internal control for protein loading and transfer efficiency.