4.3. Von Kossa Staining

The rats’ thoracic aortas were fixed in 4% paraformaldehyde and embedded in paraffin blocks. The sections were stained for VC using von Kossa’s method. Briefly, the sections were deparaffinized and washed with distilled water. They were then placed in 5% silver nitrate solution, exposed to sunlight for 20 min, and rinsed with distilled water. The sections were subsequently placed in 5% sodium thiosulfate for 5 min and ten washed with distilled water. Finally, they were counterstained with nuclear fast red for 5 min. Dark brown staining indicated areas of calcification in the vessel walls. Calcification was evaluated semi-quantitatively using Image J software (National Institutes of Health, Bethesda, MD, USA). The percentage of calcified area was calculated as the von Kossa–positive area normalized to the total tissue area, which was measured in 10 representative areas and expressed as a mean value.