TSC2 deletion in macrophages causes iron deficiency. (A-B) Representative image showing ferric iron staining using Prussian blue, counterstained with nuclear fast red in the BM (A) and on splenic formalin-fixed paraffin-embedded (FFPE) sections (B) of +/+ and cre/+ mice. Scale bar, 100 μm. (C) The total iron concentration was measured via an atomic absorption spectrometry (AAS) using the spleen of control (+/+) and cre/+ mice aged between 8 and 12 weeks old (∗∗∗P < .001). Total iron levels were normalized with respect to the tissue weight. (D) Representative image of immunofluorescent staining for ferritin (iron storage protein) and F4/80 (macrophage marker) on mouse spleen cryosections. Scale bar, 50 μm. (E) Total serum iron concentration was measured via AAS. (F) Total serum heme concentration was measured spectrometrically. (G) Tf saturation was determined in serum of control (+/+) and cre/+ mice (∗P < .05). (H) Total Tf concentration in the serum was determined using an enzyme-linked immunosorbent assay. (I-J) The TIBC (I) and unsaturated iron binding capacity (J) of Tf in the serum from control (+/+) and cre/+ mice were determined (∗∗ P < .01). (K) Steady-state levels of the Fe-free Apo-Tf (Apo), monoferric-Tf (Mono), and Fe-saturated holo-Tf (Holo) in control (+/+) and cre/+ mice as visualized on urea gel electrophoresis. Human apo-Tf and holo-Tf (hTf) were used as controls (lane1).