Excess circulating apo-Tf enhances dietary iron absorption, whereas injected fluorescent-conjugated transferrin colocalizes with lamina propria macrophages. (A) Schematic experimental outline. Male 7- to 8-week-old wild-type (C57BL/6) mice (n = 6-10 per group) were fed an IDD (2-6 ppm iron) for 1 week. Afterwards, they were switched to an HID (0.25% carbonyl iron) and/or injected intravenously (tail vein) with 10 mg apo-Tf or holo-Tf. After 6 hours, the animals were sacrificed; serum was prepared and used for analysis of iron indexes; duodenal sections were prepared and used for histologic analysis of iron. (B) Serum TBI. (C) Transferrin saturation. (D) Histologic detection of iron deposits in enterocytes by Perls staining (black arrowheads); a section from a mouse on standard diet was used as control. (E) TIBC. (F) NTBI. (G) Rosa26^mT-mG/+;LysM-Cre reporter mice were generated by breeding Rosa26^mT-mG/+ (see Charlebois et al²) and LysM-Cre animals. The reporter mice were injected with 10 mg apo-Tf spiked with 100 μg Alexa Fluor 647 (AF647)–conjugated transferrin. After 30 minutes, the animals were sacrificed, and tissues were collected. Frozen liver (top) and duodenal (bottom) sections were analyzed by fluorescent confocal microscopy; DAPI (4′,6-diamidino-2-phenylindole) staining was used for nuclear visualization. Signal corresponding to mT (membrane-targeted tandem dimer Tomato) is shown in red; DAPI in blue, mG (membrane-targeted enhanced green fluorescent protein) in green, and AF647-Tf in pink. Merged images are shown on the right panels; white arrowheads indicate colocalization of AF647-Tf with green macrophages. Scale bars, 20 or 10 μm. Serum data (B-E) are represented as mean ± SEM. Statistical differences (P < .05) were determined using 1-way analysis of variance, except the comparison between “-” and “apo-Tf” groups (B), which was done by the unpaired Student t test. ∗∗P < .01, ∗∗∗P < .001.