*2.5. Histochemical Determination of Iron in the Tissues*

Tissues of the liver, left heart ventricle, kidneys and aorta of control and USPION-treated rats were collected and routinely fixed at 10% buffered neutral formalin. Samples were routinely fixed in paraffin and then cut into 5 μm slices. Slices were deparaffinised and the Perl's method for iron staining was used for determination of iron in the tissues of USPION-treated and control rats as described previously [28]. Iron was converted to ferric ion with acid solutions of ferrocyanides. Ferric iron in the tissue react with the ferrocyanide, resulting in the formation of a blue pigment called Prussian blue. Nuclei are stained in red by safranine method.
