Tunel staining kit

To detect apoptosis in the collected ovaries, terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) staining kits (Roche Applied Science, USA) were used according to the manufacturer’s instructions. The nuclei were counterstained using DAPI. Images were collected under a fluorescence microscope (Olympus). The percentage of TUNEL-positive cells was determined by counting five random fields from each sample. The results are expressed as the percentages of apoptotic cells in each section.

The reagent contained mangiferin, doxorubicin, dimethyl sulfoxide (DMSO), SDS, tris, glycine, BSA, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenylTrazolium Bromide (MTT), and CelLytic (Sigma-Aldrich, St. Louis, MO, USA). Other ingredients included Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antimycotic/antibiotic mixtures (Carlsbad, CA, USA), DCFH2-DA, and TUNEL staining kits (Roche, Mannheim, Germany), and antibodies were purchased from Invitrogen, Carlsbad, CA, USA. Antibodies to AKT, pPI3K, PI3K PERK1/2, ERK1/2, pP38, P38, BCL2 PARP, HO-1, NQO1, Keap-1, pNrf2, COX, TNF-α, pNFC-B, β-actin, and Lamin B1 were sourced from Invitrogen and Thermo Fisher, Waltham, MA, USA. The antibody against cleaved caspase-3 was purchased from Abcam (Branford, CT, USA).

Rats were euthanized 24 h after SAH and perfused with PBS (0.1 mol/L) and paraformaldehyde (4%, pH 7.4) via the heart. The brain was then removed and fixed by immersing in 4% paraformaldehyde (pH 7.4) at 4°C for 24 h, followed by dehydration with 30% sucrose for 72 h. Coronal slices were made of the brain tissue (7-μm-thick) using a cryostat sectioning machine. Coronal slices were washed with 0.01 M PBS and then blocked with a blocking buffer (10% goat serum, 0.1% Triton X-100). The primary antibody was rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:100, ab178847, Abcam, United States). The slices were washed with PBS and incubated with rhodamine-coupled goat anti-rabbit antibody (1:200, Jackson Immunoresearch, United States) at 25°C in the dark for 2 h. Apoptotic cell death was analyzed using a TUNEL staining kit (Roche Inc., Basel, Switzerland). The TUNEL staining procedure was performed strictly in accordance with the manufacturer’s instructions. The slices were rinsed and stained with 4′,6-diamidino-2-phenylindole (DAPI) and fixed in glycerol. Three sections around the bifurcation were used for each rat and three random fields (×40) in the ipsilateral basal cortex were acquired under a fluorescence microscope (Olympus, Tokyo, Japan), and the images were analyzed using Image Pro Plus by a blinded investigator.

To investigate apoptosis, TUNEL staining of heart tissues and primary cardiomyocytes was performed according to the protocol of TUNEL Staining Kit (Roche, USA). Cell nuclei labeled with 4,5-diamino-2-phenylindole (DAPI) appeared in blue color, and apoptotic nuclei labeled with TUNEL- positive staining were detected by red fluorescent staining.

A TUNEL staining kit was used to detect DNA strand breaks according to the instructions provided by Roche Molecular System (Branchburg, NJ). The number of TUNEL‐positive nuclei per field (n = 10) was evaluated.