Quantification of Myocardial Fibrosis and Apoptosis

Histological analysis of fibrosis, and cell surface area measurements were described previously [25 (link)]. Briefly, mouse hearts (N = 4–5) were fixed in 10% formalin/phosphate-buffered saline (PBS) and dehydrated for paraffin embedding. The dehydrated, paraffin-embedded fixed hearts were then cut into 5 μm sections for Masson’s trichrome staining. The percentage of myocardial fibrosis was determined using the ratio of the total interstitial fibrosis area to longitudinal sectional area of a left ventricle section using MetaMorph 6.1 software (Molecular Devices, LLC., San Jose, CA, USA) as described previously [25 (link)]. Using a section of dehydrated, paraffin-embedded fixed hearts, TUNEL was performed using TMR Red in situ cell death detection kit following the manufacturer’s instructions.