Hearts were fixed in 4% (wt/vol.) paraformaldehyde, embedded in paraffin, sectioned at 5 μm, and stained with Sirius Red F3BA (0.5% wt/vol. in saturated aqueous picric acid; Aldrich Chemical Company, St Louis, MO, USA) for the measurement of collagen volume fraction. Cardiac interstitial fibrosis and the ratio of wall to lumen area of coronary artery were quantified, as described [34 (link)]. The positive area of fibrosis per field area was assessed by examining at least 10 fields per rat using Lumina Vision version 2.2 analysis software.
For ED-1 immunohistochemistry, cardiac sections were incubated overnight with ED1 antibody (×100; BMA Biomedicals, Switzerland) followed by Histofine simple stain Max-Po (M) (Nichirei, Biosciences, Tokyo, Japan). The number of cardiac ED-1-positive cells per mm2 was counted in a blinded manner by examining more than 10 fields per section using a microscope with X200 magnification. The average ED-1-positive cell number was obtained in each rat.
For ED-1 immunohistochemistry, cardiac sections were incubated overnight with ED1 antibody (×100; BMA Biomedicals, Switzerland) followed by Histofine simple stain Max-Po (M) (Nichirei, Biosciences, Tokyo, Japan). The number of cardiac ED-1-positive cells per mm2 was counted in a blinded manner by examining more than 10 fields per section using a microscope with X200 magnification. The average ED-1-positive cell number was obtained in each rat.
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