Axiovision image analysis software

At sacrifice, aorta, left femoral, and left carotid arteries were isolated. For histological analyses, thoracic aortae were fixed in neutral buffered formalin for 90 min and cut into 2–3 mm thick rings. These rings were then embedded upright in paraffin, of which 4 μm sections were cut and stained for calcification with von Kossa and counterstained with hematoxylin and eosin (H&E). Calcification was evaluated using Axiovision image analysis software (Release 4.5, Carl Zeiss, Oberkochen, Germany). Two color thresholds allowed separation of the calcified area (von Kossa positive, black areas) and the remaining tissue area. The percentage calcified area is defined as the von Kossa positive area versus the total tissue area (von Kossa positive area plus the remaining tissue area).
To determine the calcium content, the proximal part of the abdominal aorta, the left carotid, and femoral arteries were weighed on a precision balance. The tissue samples were then digested overnight in 65% HNO₃ at 60 °C, followed by dilution in 0.1% La(NO₃)₃ to eliminate chemical interference during flame atomic absorption spectrometry. Results were expressed as mg calcium/g wet tissue.

Six diseased human coronary arteries and five control internal thoracic arteries were harvested from five patients with end-stage heart failure undergoing heart transplantation at Tri-Service General Hospital in Taipei, as approved by the Hospital Human Subjects Review Committee (IRB No.: 1–103–05-132). Samples were fixed and embedded in paraffin blocks for immunohistochemical studies. Three serial sections were performed for phospho-Smad2, FN, and LM, respectively, and were counterstained with the EC marker von Willebrand factor (vWF) and the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI). Briefly, serial sections (5 μm thick) of human coronary and internal thoracic arteries were placed on poly-L-lysine-coated slides, deparaffinized, and blocked for 1 h with PBS containing 5 mg/mL of serum albumin. One section was incubated with anti-phospho-Smad2 antibody (1:50) for 1 h at 37 °C, followed by rhodamine-conjugated secondary antibody (1:100) in the presence of DAPI for 2 h at room temperature. The second and third sections were incubated with an antibody (1:50 each) against laminin or fibronectin, respectively, followed by the secondary antibodies for 2 h at room temperature. Images were acquired and analyzed using a Zeiss fluorescence microscope with Axiovision image analysis software.

Arterial calcifications were quantified via determining (i) the calcium content in the abdominal aorta and smaller arteries by use of FAAS method and (ii) percentage calcified area on Von Kossa stained sections of the thoracic aorta. First, the abdominal aorta, carotid and femoral arteries were weighed on a precision balance where after the samples were digested in 65% HNO₃ at 60 °C for 6 h. After appropriate dilution in 0.1% La(NO₃)₃ to eliminate chemical interference, the calcium content in the digests was measured with FAAS and expressed as mg calcium/g wet tissue. Second, the thoracic aorta was fixed in neutral buffered formalin for 90 min. Approximately, 15–20 tissue parts of 2 to 3 mm were cut and embedded upright in a paraffin block. Aortic sections (4 µM thick) were stained with Von Kossa and counterstained with hematoxylin and eosin. Axiovision image analysis software (Release 4.5; Carl Zeiss, Oberkochen, Germany) was used to calculate the percentage calcified area, which equals the ratio of pixels of Von Kossa positive area versus the pixels of total tissue area.

The left tibia was fixed in 70% ethanol overnight at 4°C, dehydrated, and embedded in 100% methylmethacrylate (Merck, Hohenbrunn, Germany) for bone histomorphometry. For analysis of static bone parameters, 5-μm-thick sections were Goldner stained for visualization and measurement of total bone area, mineralized bone area, osteoid area, eroded perimeter, and osteoblast and osteoclast perimeter using the Axiovision image analysis software (release 4.5; Carl Zeiss).

After isolation of the thoracic aorta of the adenine-induced CRF rats, tissue was fixed in neutral buffered formalin and cut into sections of 2–3 mm. These sections were embedded upright in a paraffin block and a 4 µm section was stained for vascular calcification with Von Kossa's method and counterstained with haematoxylin and eosin. The% of calcified area was calculated using Axiovision image analysis software (Release 4.5, Carl Zeiss, Oberkochen, Germany) in which two color separation thresholds measure the total tissue area and the Von Kossa positive area. After summing both absolute areas, the% of calcified area was calculated as the ratio of the Von Kossa positive area versus the total tissue area.
The proximal part of the abdominal aorta was isolated and weighed on a precision scale. Subsequently, samples were digested in 65% HNO₃ at 60°C overnight. The calcium and magnesium content of the tissue was measured with flame atomic absorption spectrometry and expressed as mg calcium or magnesium/g wet tissue.

To visualize and quantify calcification in the aortic wall, sections of the thoracic aorta were stained using Von Kossa’s method. Following isolation, the thoracic aorta was fixed in neutral buffered formalin for 90 minutes. It was then cut into 15–20 segments of 2 to 3 mm which were embedded upright in a paraffin block. Four-micrometer-thick sections were stained with Von Kossa’s and counterstained with hematoxylin and eosin to detect and quantify calcification. The percentage of calcified area was calculated using Axiovision image analysis software (release 4.5; Carl Zeiss, Oberkochen, Germany), in which 2 color-separation thresholds measure the total tissue area and the Von Kossa positive area. After summing both absolute areas, the percentage of calcified area was calculated as the ratio of the Von Kossa positive area versus the total tissue area. Calcification in the aorta and smaller arteries was also quantified by measurement of the calcium content. The proximal part of the abdominal aorta and the left carotid and femoral arteries were isolated and directly weighed on a precision balance. The arterial samples were digested in 65% HNO₃ at 60°C for 6 hours. The calcium content in the digested samples was measured with flame atomic absorption spectrometry and expressed as milligrams of calcium/gram of wet tissue.