Bx51 dsu

TUNEL analysis was performed to measure the degree of tissue apoptosis using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer's instructions. Frozen free-floating brain sections were visualized with a confocal microscope (BX51-DSU, Olympus) and digital images captured and documented. TUNEL-positive cells were manually counted in the CA3 region (100×100 µm) in three sections (n=4 per group). The cells were counted by observers blinded to the treatment conditions using 20× objectives.

TUNEL was performed on frozen tissue sections using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's protocol. The sections were examined under a microscope (BX51-DSU, Olympus, Tokyo, Japan), and digital images were captured. For each treatment group, TUNEL-positive cells were manually counted in the CA3 region (50×50 µm) in three sections (n=5–7 mice/group). The cells were counted by observers who were blinded to the treatment conditions.

Free-floating brain tissue sections were incubated with two antibodies: mouse anti-glial fibrillary acidic protein (GFAP, Santa Cruz Biotechnology, Dallas, TX, USA; 1:500) antibody; goat anti-LCN2 (Abcam, Cambridge, UK; 1:200) antibody; rabbit anti-Iba-1 (Wako Pure Chemical Industry Richmond, VA, USA; 1:200). Samples were washed with 0.1 M PBS and incubated with Alexa Fluor 488-conjugated donkey anti-mouse or 594-anti-goat secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. The sections imaging were carried out using a microscope and captured (BX51-DSU, Olympus), three consecutive sections per animal were analyzed and similar sections were used.

Cells were grown on glass cover slips in 24-well culture plates at 2×10⁴ cells per well for 24 hours. Then, the cells were treated with 50 µM of quercetin for 24 hours. Next, cells were washed with phosphate buffered saline (PBS), fixed for 15 minutes at room temperature with 4% paraformaldehyde, and permeabilised for 15 minutes with 0.3% Triton X-100 in PBS for 10 minutes. Then, the cells were blocked with 5% normal donkey serum in PBS for 1 hour. Subsequently, the cells were incubated at 4℃ with the desired primary antibody in 5% normal donkey serum overnight, followed by incubation with a specific fluorescence-conjugated secondary IgG (Invitrogen) for 1 hour in the dark. Finally, cells were washed with PBS and mounted using ProLong Gold antifade mountant with 4′,6-diamidino-2-phenylindole dichloride (DAPI) (Invitrogen) for nuclear staining. All images were taken using a fluorescence microscope (BX51-DSU, Olympus, Tokyo, Japan).

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) staining was performed to detect cells death using the In Situ Cell Death Detection Kit, TMR Red (Roche Molecular Biochemicals, Mannheim, Germany). Cells were grown on glass coverslips in 24-well culture plates at 2×10⁴ cells per well for 24 hours. Next, the cells were treated with 50 µM of quercetin for 24 hours. Cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and permeabilised for 2 minutes with 0.5% Triton X-100 in PBS on ice. Cells were then washed in PBS and incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT), and the reaction buffer containing fluorescein-dUTP (Roche Applied Sciences, Mannheim, Germany) for 60 minutes at 37℃. Finally, cells were washed with PBS and mounted using Pro-Long Gold antifade mountant with DAPI for nuclear staining. All images were taken using a fluorescence microscope (BX51-DSU, Olympus).