Anti gap43

Total protein was extracted from transfected OGD/R-treated cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA assay kit (Beyotime Institute of Biotechnology) and protein samples (20 µg/lane) were separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% non-fat milk at room temperature for 1 h. The membranes were then incubated with the following primary antibodies overnight at 4°C: Anti-endothelial nitric oxide synthase (eNOS; 1:1,000; cat. no. ab5589; Abcam), anti-MAP-2 (1:1,000; cat. no. ab32454; Abcam), anti-GAP-43 (1:1,000; cat. no. ab75810; Abcam), anti-Bcl-2 (1:1,000; cat. no. ab196495; Abcam), anti-Bax (1:1,000; cat. no. ab182733; Abcam), anti-cleaved caspase-3 (1:5,000; cat. no. ab214430; Abcam), anti-caspase-3 (1:2,000; cat. no. ab184787; Abcam) and anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). Following the primary antibody incubation, the membranes were washed twice with TBS with 0.05% Tween-20 (TBST) and incubated with goat anti-rabbit IgG H&L secondary antibody (1:2,000; cat. no. ab6721; Abcam) for 1 h at room temperature. GAPDH was used as the internal loading control. Protein bands were visualized using ECL substrate (Pierce; Thermo Fisher Scientific, Inc.) and analyzed with ImageJ software (version 3.0; National Institutes of Health).

The right hemisphere of APP/PS1 mice was fixed in 4% paraformaldehyde (Guoyao) and embedded with paraffin. Coronal paraffin sections (6-μm thick) were dewaxed, rehydrated, and treated with 3% H₂O₂/methanol solution for 10 min. Then, sections were boiled for 15 min in 0.01 M citrate buffer (pH 6.0). After the citrate buffer returned to room temperature, sections were washed with PBS and incubated in blocking solution (5% BSA) for 1 hr at 37°C, followed by being incubated overnight with primary antibodies at 4°C in a humidified chamber. Primary antibodies included anti-synaptophysin (1:250, Abcam) and anti-GAP43 (1:250, Abcam) rabbit antibodies and mouse monoclonal anti-Aβ antibody (6E10, 1:500, Covance). After being washed with PBS, the sections were incubated with anti-mouse or rabbit IgG-horseradish peroxidase (HRP) secondary antibody for 1 hr at 37°C, followed by diaminobenzidine (DAB) (Boster Biotech) and hematoxylin staining. Slides were assessed by light microscope (IX71, Olympus) and micrographs were analyzed by Image-Pro plus 6.0 software.

After incubation under different experimental conditions for 24 hours, double fluorescence labeling for GAP-43 and MAP2 expression was performed using rabbit polyclonal anti-GAP-43 (1:500; Abcam, Cambridge, MA, USA) or mouse monoclonal anti-MAP2 (1:400; Abcam) as the primary antibodies. The secondary antibodies were goat anti-rabbit conjugated to Cy3 (1:200; Abcam) or goat anti-mouse conjugated to Cy2 (1:200; Abcam). All procedures were performed in the dark to preserve fluorescence. Before incubation with the primary antibody, the cells were processed for fixation, non-specific site blocking, and permeabilization. Three washes with 0.1 M phosphate buffered saline were carried out between each step before covering with a coverslip. Slide observation and image capture were performed under a fluorescent microscope (Olympus BX63, Olympus Corporation, Tokyo, Japan) with a digital photo processing system (cellSens Dimension, version 1.6, Olympus Corporation, Tokyo, Japan).