Changes in iron distribution in PR65-treated hepcidin knockout mice. Tissue iron was visualized by enhanced Perls stain at 0 to 48 hours after subcutaneous injection of PR65 (100 nmol). Representative images are shown. Horizontal bars indicate 400 μm (10×) and 100 μm (40×). Top row: Spleen iron was scant and its distribution did not change appreciably during the 48 hours. Middle row: Iron in the villus stroma was evident in solvent-treated and 1 to 4 hours PR65–treated mice, indicating active ferroportin-mediated efflux of iron from basolateral membranes of enterocytes. From 12 to 24 hours, iron was retained in enterocytes consistent with (mini)hepcidin-induced ferroportin degradation. Forty-eight hours after injection iron was no longer retained by enterocytes. Bottom row: As expected, the livers were iron-loaded at baseline and no changes in the pattern of iron staining were seen within 48 hours of PR65 treatment.