Protein expression of iron metabolism genes in liver, spleen, and duodenum of anemic rats. Rats were injected with PG-APS injection and/or phlebotomized as detailed in “Animals.” Protein extracts from liver (A) and spleen (B) were prepared from nitrogen snap-frozen tissue obtained from control, bleeded (IDA), ACD, and ACD/IDA rats and run on a 10% to 15% sodium dodecyl sulfate–polyacrylamide gel as detailed in “Western blotting.” One of 4 representative experiments is shown. Bright-field photographs of rat duodenum immunohistochemistry (C) were taken from control (i), phlebotomized (IDA; ii), ACD (iii), and ACD/IDA (iv) rats using affinity-purified antiferroportin antibody. ↑ indicates the basolateral ferroportin expression. The images were captured by a Olympus microscope BX51 (Olympus Plan FL20 × /0.75; Olympus, Hamburg, Germany) using a ProgResC12 plus camera (Jenoptik, Jena, Germany) and ProgResCapturePro2.5 software. One of 4 representative slides is shown.