Deadend fluorometric terminal

A DeadEnd Fluorometric terminal deoxynucleotidyl transferase–mediated dutp nick end labeling (TUNEL) System Kit (Promega, G3250) was used for the TUNEL assay according to the instruction. Briefly, the tissue sections were deparaffinized in fresh xylene and graded ethanol and fixed by immersing the slides in 4% methanol-free formaldehyde. After washing twice with PBS buffer, proteinase K (20 μg/ml) was added to each slide to cover the tissue section for 10 min at room temperature. Subsequently, the tissue sections were fixed with 4% methanol-free formaldehyde for 5 min. After washing with PBS buffer, the slide was covered with equilibration buffer for a couple of minutes, followed by incubation with Terminal Deoxynucleotidyl Transferase, Recombinant buffer at 37 °C for 1 h in the dark. After terminating the reactions by immersing the slides in 2× saline sodium citrate for 15 min at room temperature followed by washing with fresh PBS buffer three times to remove unincorporated fluorescein-12-2'-deoxyuridine 5'-triphosphate (dUTP), cells were stained with 0.1 μg/ml 4,6-diamino-2-phenyl indole for 10 min at room temperature. The slides were sealed with an antifluorescence quenching reagent, and images were captured using a confocal microscope.

Cell viability was determined by using the CellTiter-Glo luminescent cell viability assay (Promega Corporation, Madison, WI, USA) according to the manufacturer’s directions. DNA fragmentation was determined by using the DeadEnd™ Fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System (Promega Corporation) according to the manufacturer’s directions. Nuclei were stained using Vectasheild mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA).

Hearts were perfused with 10% formalin, dehydrated on an Autotechnicon Mono Tissue Processor (Technicon Corporation), embedded in paraffin with a Leica HistoEmbedder, and sectioned using a Leica RM2255 Microtome. Sections were stained with hematoxylin and eosin and Masson's trichrome according to the manufacturer's protocols. Apoptosis detection was performed using the Promega DeadEnd Fluorometric terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) system according to manufacturer's instructions. Hematoxylin and eosin and Masson's trichome images were collected on a Nikon eclipse with a Nikon DS‐Qi1Mc camera. TUNEL images were collected on a Zeiss LSM 700 confocal. Images were analyzed using Image J (NIH) by a group‐blinded researcher.