The animals were sacrificed 3 days or 3 weeks after AMI. The tissues were fixed in formalin and embedded in paraffin blocks according to established protocols. The fixed hearts were serially cut at 8 µm from the apex to the level just below the coronary artery ligation site. After antigen retrieval, the specimens were incubated with 1% normal blocking serum in PBS for 60 min to suppress the nonspecific binding of IgG. The slides were then incubated for 30-60 min with each mouse antibody or fluorescent reagent.
Three days after the AMI, the infarcted tissue was stained with EdU (Invitrogen) to identify the live MSCs, and the TUNEL kit (R&D Systems) was used to identify the nuclei of the apoptotic cardiac myocytes in the infarct border zone. Three weeks after the AMI, the infarct border zone was stained with Texas Red-X-conjugated wheat germ agglutinin (WGA, Invitrogen) and FITC-conjugated CD31 (BD Biosciences) to measure the vessel density. The tissue was further stained with anti-myosin heavy chain eFluor 660 (eBioscience) and 4, 6-diamidino-2-phenylindole (DAPI, Roche) to measure the cardiac myosin-positive area in the infarct zone with Image Pro Plus software. Fluorescence microscopy (Olympus BX61) or a confocal laser scanning (CLS) microscopy system (Thorlabs, Inc.) was used as necessary to obtain images of the immunostaining results.
Three days after the AMI, the infarcted tissue was stained with EdU (Invitrogen) to identify the live MSCs, and the TUNEL kit (R&D Systems) was used to identify the nuclei of the apoptotic cardiac myocytes in the infarct border zone. Three weeks after the AMI, the infarct border zone was stained with Texas Red-X-conjugated wheat germ agglutinin (WGA, Invitrogen) and FITC-conjugated CD31 (BD Biosciences) to measure the vessel density. The tissue was further stained with anti-myosin heavy chain eFluor 660 (eBioscience) and 4, 6-diamidino-2-phenylindole (DAPI, Roche) to measure the cardiac myosin-positive area in the infarct zone with Image Pro Plus software. Fluorescence microscopy (Olympus BX61) or a confocal laser scanning (CLS) microscopy system (Thorlabs, Inc.) was used as necessary to obtain images of the immunostaining results.