Necroptosis Assay

Myocardium necroptosis was measured by EBD-Caveolin 3 (CaV3) double staining. Mice were intraperitoneally given EBD (10 mg/ml) 14 h before the MI/R surgery. After surgerical operation, excised hearts were put into optimal cutting temperature (OCT) compound and were sliced into 5-μm cryosections. Finally, sections were incubated with CaV3 antibody and were imaged using a fluorescence microscope (Olympus, Tokyo, Japan).

Propidium iodide (PI)/Annexin V staining was used to distinguish the necroptotic cells. V-ZAD-FMK (Selleck Chemicals, TX, United States) was added to inhibit caspases accompanied with vehicle or 4-HNE, then the cells were harvested and washed twice. Cells were resuspended at the concentration of 1 × 10⁶/ml and then were incubated with PI and Annexin V at 37°C for 20 min. The cells were detected using flow cytometry and were analyzed with CytExpert software (Beckman Coulter, IN, United States).