*3.3. Histochemical Staining of Iron in the Tissues*

The samples of the liver, left heart ventricle, kidneys and aorta of control and USPION-treated rats stained using the Perl's method confirmed the presence of iron in the tissues of control and USPION-treated rats. However, it is not possible to unequivocally confirm that iron content in USPION-treated samples is due to USPIONs presence in these tissues (Figure 18).

**Figure 18.** Iron staining in the liver, left heart ventricle, kidneys and aorta of control (left column) and USPION-treated (right column) rats. Staining of iron in the spleen of control rat (bottom left, positive control of biogenic iron) and positive staining of iron in the liver of rats treated with USPIONs at the dose of 20 mg Fe/kg (bottom right, positive control of USPION-originated iron). Prussian blue pigment determines iron. Nuclei are stained in red.
