In situ cell death detection kit tunel fluorescence fitc kit

Apoptosis of cardiomyocytes was detected by staining ventricular specimens from border zone (3 days post-MI) and neonatal rat cardiomyocytes (4 hours after 100 μM H₂O₂) with the In Situ Cell Death Detection Kit (TUNEL fluorescence FITC kit, Roche) according to manufacturer's protocols. After TUNEL staining, the NRVMs or ventricular specimens were immerged into the DAPI (Sigma-Aldrich) solution to stain the nuclei of living and apoptotic cells. Laser scanning confocal microscope (Olympus, Fluoview1000, Tokyo, Japan) was used to view the fluorescence staining.

TUNEL assay with the In Situ Cell Death Detection Kit (TUNEL fluorescence FITC kit, Roche, Indianapolis, IN, U.S.A.) was used to evaluate DNA fragmentation of individual cells. Cells cultured on coverslips were washed with PBS containing 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄ and 1.4 mM KH₂PO₄, pH 7.4, followed by fixation in 4% paraformaldehyde solution (PFA) for 1 h at 4°C. Cells were then permeabilized using 0.1% Triton X-100 solution for 2 min, and cells were incubated in freshly prepared TUNEL reaction mixture for 1 h at 37°C in dark. The coverslips were then washed with PBS. Following this, the coverslips were mounted on slides with Prolong anti-fade solution (Invitrogen, U.S.A.) and TUNEL staining was analyzed with a fluorescence microscopy (Eclipse 80i; Nikon Co., Tokyo, Japan).