Application suites v4

Segments from proximal epicardial arteries (2–4 mm in length) were placed in 10% phosphate-buffered formalin for 24–48 h, then transferred to 70% ethanol. The proximal epicardial arteries used for histology and intracellular Ca²⁺ measures were not bypassed previously. We assume that blood flow never was zero even in the ischemic arteries defined by the >75% plaque burden. Histology of arterial cross-sections was performed in the Department of Anatomy and Cell Biology at Indiana University School of Medicine. Verhoeff-Van Gieson elastin stain was used to determine media area and plaque burden, which we define as the percentage of the original lumen that is occupied by atherosclerotic plaque. Von Kossa stains calcified areas black to determine vascular calcification. Masson’s Trichrome stain was used to visualize collagen (blue) and cellular composition (red). Images were captured with a Leica DM3000 microscope connected to Leica Application Suites V4.1 software (Leica Microsystems GmbH, Wetzlar, Germany) and analyzed using Adobe Photoshop^® CS6.

Coronary artery segments were collected at euthanasia and fixed in 10% phosphate buffered formalin for 24 hours before being switched to 70% ethanol solution. Samples were paraffin embedded, sectioned (5 μm thick), mounted on a slide and stained by the Indiana University Histology Core (Indiana University School of Medicine, Indianapolis, IN). Verhoeff-Van Gieson (VVG) elastin staining was performed to determine the location of the external elastic lamina (EEL) and the internal elastic lamina (IEL) to assess wall thickness, medial area, and the intima/media ratio as measures of atherosclerosis progression (50 (link), 54 (link)). Wall thickening (percent media plus percent neointima) was measured by the following equation:
$$\left[\left(\frac{\mathit{Area}\mathit{within}\mathit{EEL}-\mathit{Area}\mathit{within}\mathit{IEL}}{\mathit{Area}\mathit{within}\mathit{EEL}}\right)+\left(\frac{\mathit{Area}\mathit{within}\mathit{IEL}-\mathit{Area}of\mathit{Lumen}}{\mathit{Area}\mathit{within}\mathit{EEL}}\right)\right]\times 100\%$$
The media area was calculated as the area within the EEL minus the area within the IEL. The intima area was calculated as the area within the IEL minus the area of the lumen. All images were captured with a Leica DM3000 microscope connected to Leica Application Suites V4.1 software (Leica Microsystems GmbH, Wetzlar, Germany) and analyzed using (Adobe Photoshop® CS6).