Dapi staining solution

Gastric cancer cells (1 × 10⁵) were inoculated into 24‐well plates and incubated for 24 h at 37°C. The cells were then fixed for 15 min by 4% paraformaldehyde, followed by cell permeation using PBS supplemented with 0.3% Triton X‐100 for 15 min. The cells were subsequently blocked for 60 min by PBS containing 3% BSA, after which they were incubated for 1 h with cortactin primary Abs (ab81208; Abcam) at 37°C. This was followed by 1 h of incubation in the dark with the secondary Abs (ab150113; Abcam) at 37°C as well as a 1 h incubation in the dark with phalloidin (Solarbio) at room temperature. The cells were then incubated in the dark with DAPI staining solution (Beyotime) for 10 min at room temperature. Finally, they were transferred to slides prior to visualization and the capture of immunofluorescence images with an FV3000 confocal laser scanning microscope (Olympus).

Following various treatments and interventions, all groups of cells were washed with PBS for 5 min. The cells were then fixed with 4% paraformaldehyde for 15 min, blocked with a blocking solution containing 10% calf serum for 45 min at RT, incubated with anti-NAMPT antibody (dilution, 1:200; cat no. ab45890; Abcam, Cambridge, MA, USA) at 37°C for 1 h, and placed in a 4°C refrigerator overnight. After washing with PBS, the cells were incubated with fluorescein isothiocyanate-labelled goat anti-rabbit IgG (dilution, 1:500; cat no. A0562; Beyotime Institute of Biotechnology) for 1 h in a 37°C incubator. After 3 rinses with PBS, the cells were covered with DAPI staining solution (cat no. C1005; Beyotime Institute of Biotechnology) and incubated at RT for 5 min. The cells were rinsed 3 more times with PBS (5 min each time). Subsequently, the cells were mounted onto glass slides in PBS and glycerol (1:1). The fluorescence signal was examined using an Olympus BX51 biological microscope at a magnification of ×200.

Tissue sections were sequentially incubated in xylene and different concentrations of ethanol. Antigen retrieval was performed using microwave treatment, followed by blocking with 5% BSA at room temperature for 60 minutes. Subsequently, the sections were incubated overnight at 4°C with an appropriately diluted primary antibody. After the tissue sections were rewarmed the next day, they were incubated with an appropriately diluted secondary antibody specific to the corresponding species (Proteintech, Wuhan, China) at room temperature in the dark for 60 minutes. Finally, nuclear staining was performed using DAPI staining solution (Beyotime, Shanghai, China), and the slides were sealed with anti-fade mounting medium to prevent fluorescence quenching. Samples were analyzed using a fluorescence microscope (Nikon, Tokyo, Japan), and microscopic images were obtained.

The combination of ketamine and chlorpromazine can be used for anesthesia in rats (35 (link),36 (link)). In the present study, the rats (n=6/subgroup) were anesthetized with a combination of ketamine (100 mg/kg) and chlorpromazine (5 mg/kg) via intra-peritoneal injection, according to our previous studies (37 (link),38 (link)). Following deep anesthesia determined by respiratory, palpebral reflex, pedal withdrawal reflex, and cutaneous reflex (39 (link),40 (link)), the animals were transcardially perfused with 400 ml of saline followed by 4% paraformaldehyde solution (pH 7.2-7.4). Following perfusion, the brain was dissected from the skull, and the auditory cortex was separated and immersed overnight in the same fixative. The right side was prepared for TUNEL staining and the left side was prepared for immunofluorescence. The following processes were performed, as previously described (41 (link)). Apoptosis in the auditory cortex of rats was detected using a TUNEL assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol. DAPI staining solution (1 µg/ml; Beyotime Institute of Biotechnology, Haimen, China) was used to counterstain the nuclei. The labeled cells were detected with a laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan).