Perls staining and tissue ferroportin protein levels of liver, spleen, and duodenum sections from mice carrying the inducible hepcidin transgenes crossed with Hfe knockout (Hfe^-/-) mice and treated for 3 weeks with doxycycline. (A-B) Typical Perls staining (A) and ferroportin immunodetection (B) using anti-mouse ferroportin antibody of liver (i,iii; original magnification × 40 in panel A and × 20 in panel B) and spleen (ii,iv; original magnification × 10) sections from Hfe^-/- (i-ii) and ind/Hfe^-/- mice (ie, mice harboring both tetO-Hepc1 and rTA^LAP-1 transgenes and with Hfe^-/- genotype; iii-iv). Nonheme iron stains blue. The liver iron content for each animal is shown. (C) Duodenum (original magnification × 20) was stained for iron (ii,iv, Perls DAB staining, nonheme iron stains brown) and ferroportin (i,iii) immunodetection in Hfe^-/- (i-ii) and in ind/Hfe^-/- mice (ie, mice harboring both tetO-Hepc1 and rTA^LAP-1 transgenes and with Hfe^-/genotype; iii,iv). All microscopy was performed using a Nikon Eclipse E800 microscope equipped with 10 ×/0.45, 20 ×/0.75, and 40 ×/0.95 oil-immersion objective lenses. Digital images were captured with a Nikon DXM1200 camera, and were acquired and processed with Nikon AC7-1 2.63 software (all from Nikon France, Champigny-sur-Marne, France).