Detection of Nramp1HA and Nramp2HA proteins by immunofluorescence in nonpermeabilized cells. Mouse Nramp1 and Nramp2 cDNAs were modified by in-frame insertion of a hemagglutinin (HA) epitope (YPYDVPDYAS) into the predicted loop delineated by putative TM7 and TM8 followed by transfection into CHO cells. Surface expression was monitored by immunofluorescent detection of the HA epitope in nonpermeabilized cells with the mouse monoclonal anti–HA antibody 16B12. Control CHO cells (A), Nramp2HA-expressing CHO cell line N2-310a (C), and Nramp1HA-expressing CHO cell line N1-1816 (B,D,E,F) were grown on glass coverslips and incubated (1 hour, 4°C) with mouse anti–HA monoclonal primary antibodies (A-C; 1/50 dilution), affinity-purified rabbit polyclonal anti–Nramp1NT antibodies (E; diluted 1/50), or without primary antibody (D). Cells were then fixed, and incubated with goat anti–mouse-IgG-Cy3 (A-D; 1/200) or goat anti–rabbit IgG-rhodamine (E-F; 1/100) secondary antibodies. In panel F, N1-1816 cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 prior to addition of primary antibodies to detect both intracellular and PM-localized Nramp1HA protein. Original magnification, × 600 for all panels.