Quantifying Cardiac Fibrosis and Apoptosis

After 24–48 h in 4% paraformaldehyde, the blocks were dehydrated and embedded in paraffin, cut into 4 μm slices, heated overnight in a 60°C incubator, and then dewaxed and stained with H&E and Masson dye. One slice was chosen from each rat and was analyzed under a microscope. Masson staining for the presence of interstitial collagen fiber accumulation was a marker of cardiac fibrosis. The ratio of interstitial fibrosis to the total left ventricular area was calculated from 20 randomly selected microscopic fields in five individual sections per heart using a camera attached to an Leica DM2000 microscope, with images further analyzed by Image-Pro Plus 5.1 (Media Cybernetics, Silver Spring, MD), excluding coronary vessels and perivascular regions.
Apoptosis in cardiac tissue was determined with the DeadEnd Fluorometric TUNEL System (Promega), which catalytically incorporates fluorescein-12-dUTP at DNA strand breaks as previously described [16 (link)]. All sections were counterstained with DAPI (Molecular Probes) at a final concentration of 2 μM. Images were viewed with epifluorescence microscopy (Leica TCS SP5) within 24 hours and analyzed with Image-Pro Plus 5.1 Software (Media Cybernetics, Silver Spring, MD).