Immunohistochemical staining of DMT1 in the intestine of anemic mk/mk mice.

Tissues were fixed in Bouin solution, embedded in paraffin, and sectioned as described.11 Sections from the proximal duodenum (I1) of wild type mice on low-iron diet [+/+ (−Fe); A, B, and C], heterozygous mk/+ (D, E, and F), and homozygousmk/mk mice (G, H, and I) were immunostained with rabbit polyclonal antisera directed against the amino terminus of DMT1 (DMT1-NT; A, D, and G), or the carboxy terminus of isoform II (non-IRE containing) DMT1 (DMT1-CT; B, E, and H), or biliary glycoprotein 1 (Bgp1; C, F, and I). Sections were stained with DAB, counterstained with methylene blue, and photographed at a magnification of 400 ×. Arrows in (A) show intense DMT1 staining at the brush border (arrows) and intracellularly in the apical region of villus enterocytes (arrowhead). In mk/+ mice (D), DMT1 is not detectable. Inmk/mk mice (G), strong DMT1 staining is apparent in the apical region of villus enterocytes (arrowhead) but not at the brush border. In (C), (F), and (I), arrows identify Bgp1 staining at the brush border (black arrows), in the supranuclear, intracellular region (white arrows) of villus enterocytes as well as in crypts cells (arrowhead). The development time for DAB was generally longer formk/+ and mk/mk (4-6 minutes) than for low-iron diet (3 minutes), indicating that the level of DMT1 expression is stronger in normal mice on low-iron diet than inmk/mk homozygotes.