Fig. 1. The distribution of kfCFTR in mitochondria-rich (MR) chloride cells in killifish opercular epithelium. Images are confocal laser scans of kfCFTR revealed by immunofluorescence of mouse anti-human CFTR and goat anti-mouse IgG Oregon Green 488 and counterstained for mitochondria with Mitotracker Red (green fluorescence + red counterstain yields yellow). (A) Membrane from a SW-adapted animal has kfCFTR immunofluorescence at 4.5 µm from the surface of the chloride cells, localized to the apical crypt (white arrows). The asterisk indicates a gap in the pavement cell layer. (B) The same frame as for A but at a depth of 7.5 µm at the plane of the nucleus of the MR cell. Blue arrows indicate the mitochondria-rich chloride cells below the apical crypt. (C) Diagram of approximate depths of the optical sections, AP, apical crypt level; CY, cytosol level. (D,E) Similar kfCFTR immunostaining, but in MR cells from fish acclimated to FW. (D) An optical section at 4.5 µm from the surface has positive kfCFTR immunofluorescence evenly distributed in the chloride cells (white arrows) as well as in the pavement cells at the plane of the pavement cell nuclei (orange arrow). (E) The same frame as for D but 10.5 µm into the tissue at the level of the MR cell nuclei. Blue arrows indicate the MR cells with diffusely distributed positive immunofluorescence for kfCFTR. (F,G) Similar immunostaining but for a fish transferred from FW to SW for 48 h. (F) An optical section 9.0 µm from the surface, with ring-shaped kfCFTR-positive immunofluorescence (white arrows); (G) 13.5 µm from the surface, with kfCFTR immunofluorescence diffusely distributed in MR cells (blue arrows). Scale bars, 20 µm.