Bh l microscope

Hearts were perfused and excised from isoflurane-euthanized mice, washed in cold PBS. The upper and middle part of the hearts were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 5 μm thickness. Collagen was quantified by masson’s trichrome staining as we described previously [38 (link)]. The blue staining represented collagen deposition. A series of 4 sections of each mouse (4 mice per group) was examined and photographed using an Olympus BH-L microscope coupled with a digital color camera. Using ImageJ software, blue-stained areas and non-stained myocyte areas from each section were determined using color-based thresholding. The percentage of total fibrosis area was calculated as the summed blue stained areas divided by total ventricular areas.
Cardiac calcium deposition was analyzed by using alizarin red staining kit (IHC WORLD, Woodstock, MD) as per the manufacturer’s protocol. Calcification area was quantified with Image J as described above.