Immunohistochemical Analysis of Cardiac Markers

The procedure and protocol have been described in our previous reports [34 (link),35 (link),36 (link)]. Re-hydrated paraffin sections were treated with 3% H₂O₂ for 30 min and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 min at room temperature. Sections were then incubated with primary antibodies against CD31 (1:100, Bio-Rad, Hercules, CA, USA), vascular endothelial growth factor (VEGF) (1:400, Abcam Inc., Cambridge, MA, USA), C-kit (1:100, Santa Cruz), GATA4 (1:500, Abcam) and SOX2 (1:100, Abcam). Three sections of heart specimens from each rat were analyzed. For quantification, three randomly selected HPFs (400× for IHC and IF studies) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was determined by summation of all numbers divided by 9.