Prolong gold antifade reagent with 4 6 diamino 2 phenylindole dapi

One or four weeks after MSC transplantation, animals were sacrificed and the hearts were dissected. From each heart, five frozen sections were prepared crossing the midlevel of the infarcted area. The sections were all fixed in ice-cold acetone and washed in PBS. Heart sections made at 1 week after transplantation were directly mounted with ProLong Gold antifade reagent with 4,6-diamino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) and the heart sections from the later stage were stained with polyclonal rabbit anti-Troponin T (TnT) antibody (Cell Signaling Tech, Boston, MA, USA) and Cy3 conjugated goat anti-rabbit IgG (Santa Cruz Biotech, Dallas, TX, USA) and then mounted with ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA). All sections were observed under a FV1000S-SIM/IX81 confocal microscope (Olympus, Tokyo, Japan). For each section, 10 high-power fields (HPF 400x) within the border zone were randomly selected and digitally photographed. Quantitative analysis of cell engraftment was performed with cellSens Entry software (Olympus, Tokyo, Japan). The integral optical density (IOD) of EGFP signal and total cell number in each microscopic field were calculated and cell engraftment was presented as the ratio of IOD and total cell number. An investigator blinded to the treatment performed the analysis.