Immunofluorescence analysis of DMT1 and Tf in RBCs.

Cells were incubated with Alexa-conjugated Tf (Alexa-Tf) before fixation and immunostaining with anti–DMT1-NT antibody and a rhodamine-conjugated secondary antibody. The slides were then examined by confocal microscopy (approximate depth of the optical sections = 1 μm), and the Alexa-TF (green; B,F) and rhodamine (red; C,G) images were overlaid (yellow identifies overlapping staining; D,H). In erythroid cell precursors (arrowhead in A and cell in E), the DMT1 staining (C,G) showed extensive overlapping staining with the distribution of Alexa-Tf (B+C = D and F+G = H; arrowhead). On the other hand, erythrocytes (arrows in panel A) were negative for both Alexa-Tf (arrows in panel B) and DMT1 (arrows in panel C) staining. Original magnification for A-D, × 400; E-H, × 1000.