Tunel staining kit

Enzyme-linked immunosorbent assay (ELISA) kits for interleukin 6 (IL-6), IL-1β, tumor necrosis factor alpha (TNFα), and 3′-nitrotyrosine (3′-NT) were purchased from Abcam PLC (#ab100712, #ab197742, #ab108910, and #ab116691, Cambridge, UK). 4-Hydroxynonenal (4-HNE) ELISA kit was obtained from Donggeboye Biological Technology Co. Ltd. (#DG30947M, Beijing, China). TUNEL staining kits were obtained from the Beyotime Institute of Biotechnology (#C1088 or #C1090, Shanghai, China). Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12), fetal bovine serum (FBS), and TRIzol were obtained from Thermo Fisher Scientific Inc. (#11320033, #10099141, #15596026, Waltham, MA, USA). PM_2.5 was collected using high-volume sampler particle collectors and the morphology, size distribution, and components of the constituents were described in a previous study [16 (link)]. All other chemicals made in China were of analytical grade.

GSH assay and TUNEL staining kits were purchased from the Beyotime Institute of Biotechnology (#S0053, #C1090 and #P0012S, Shanghai, China). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were purchased from Nanjing Jiancheng Bioengineering Institute (#C009–2 and #C010–2, Nanjing, Jiangsu, China). Elisa kits for ADMA and 4-hydroxynonenal (4-HNE) were purchased from Bio-Techne Co., Ltd. (#NBP2–66728, Minneapolis, MN, USA) and Donggeboye Biological Technology Co., LTD. (#DG30947M, Beijing, China), respectively. The Masson’s trichrome staining kit was obtained from Solarbio Science & Technology Co. LTD (#G1340, Beijing, China). Antibodies against DDAH1, cytochrome P450 2E1 (CYP2E1), glutathione S-transferase A1 (GSTA1) and β-actin were purchased from Signalway Antibody LLC (#37368, #48247, #22536, #21800, Greenbelt, MD, USA). P65, phospho-65, c-jun N-terminal kinase (JNK) and phospho-JNK antibodies were from Cell Signaling Technology (#8242, #3033, #9252, #9251, Danvers, MA, USA). APAP and dihydroethidium (DHE) were purchased from MedChemExpress LLC (#HY-66005, Monmouth Junction, NJ, USA) and Sigma Chemical Co. (#D7008, St. Louis, MO, USA), respectively. All other compounds and chemicals were of analytical purity.

TUNEL assays were carried out using a TUNEL staining kit (#C1090, Beyotime, Shanghai, China). In brief, cells were fixed in 10% formalin and permeabilized with 0.5% Triton X-100, followed by incubation with TUNEL staining reagent at 37°C in the dark for 1 hour. Additionally, nuclei were stained with Hoechst 33258 (#H3569, Thermo Fisher, CA, USA). Fluorescence images were acquired, and the apoptotic rates were calculated as the percentages of TUNEL-positive cells.

TUNEL staining was performed using a TUNEL staining kit following the manufacturer’s instructions (Beyotime, Shanghai, China). Paraffin sections were deparaffinized by immersing them in xylene, gradient alcohol, and distilled water for 2 min. After deparaffinization, the sections were washed three times with PBS. Subsequently, 20 μg/mL DNase-free proteinase K (20 mg/mL) was added dropwise, and the sections were incubated for 25 min at 25°C. Following the incubation, the sections underwent another round of washing with PBS. Then, the tissue was exposed to 50 μL TUNEL assay solution for 1 h at 37°C, with light being shielded. After this step, the sections were washed three times with PBS. Finally, the slices were sealed using an anti-fluorescence quenching-DAPI 2-in-1 sealer before being examined under a fluorescence microscope.

For immunofluorescence, the cells were fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 15 min at room temperature, and then blocked with PBS containing 5% bovine serum albumin for 1 h at room temperature. The cells were incubated with certain antibody overnight at 4 °C. After rinsing with PBS, secondary antibody was applied for 1 h at 37 °C. The slices were counterstained with DAPI. For TUNEL staining, cells were fixed and transparented as described above. Then, the cell slices were operated according to the factory instructions of TUNEL staining kit (Beyotime, China).

After the experiment, the rats were sacrificed immediately, the heart was taken, and the left ventricle was separated. The apoptosis of myocardial cells in the ischemic region in each group was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining kit (Beyotime Biotechnology) strictly according to the instructions. The sections were observed and photographed under a fluorescence microscope. TUNEL-positive cells were apoptotic cells displaying yellow-green fluorescence, while TUNEL-negative cells were normal cells without fluorescence. The apoptosis level of myocardial cells in each group was calculated.