Biovision caspglow fluorescein active caspase 3 staining kit

Apoptotic cells were analyzed by the ApopTag^® Peroxidase In Situ Apoptosis Detection Kit (Calbiochem). After treatment with TNF-α for 24 h, cells were rinsed twice in PBS before fixation for 30 min at room temperature with 4% paraformaldehyde. Next, cells were washed in PBS before incubation in the prepared solution (0.1% Triton X-100, 0.1% sodium citrate) for 5 min. Cells were then incubated with 1 TUNEL reaction mixture in a humidified atmosphere for 1 h at 37 °C in the dark, washed in PBS, and analyzed by flow cytometry. The BioVision CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Milpitas, CA, USA) was used for detection of active caspase 3.

Apoptotic cells were analyzed by the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Calbiochem). After H/R treatment, cells were rinsed twice in PBS before fixation with 4% paraformaldehyde at room temperature for 30 mins. Next, cells were washed in PBS before incubation in the prepared solution (0.1% Triton X-100, 0.1% sodium citrate) for 5 mins. Cells were then incubated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction mixtures in a humidified atmosphere for 1 h at 37 °C in the dark, washed in PBS, and analyzed by flow cytometry. The BioVision CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Milpitas, CA, USA) was used for detection of active caspase 3. For investigating apoptosis in animal cardiac tissues, tissues were soaked in 4% paraformaldehyde. Paraffin-embedded heart was cut into 2-µm-thick sections. TUNEL staining was performed for apoptosis. In brief, the tissue sections were deparaffinized in xylene, rehydrated through a graded alcohol series (100%, 90%, 85%, and 75%), and then rinsed in PBS (pH 7.2). A DNA fragmentation detection kit (FragEL; Calbiochem) was used to detect apoptotic cells in cardiac tissue sections using TUNEL assay. The numbers of positive cells were measured and averaged from three different fields of view with the heaviest TUNEL staining in the infarction zone.