Fig. 11. Immunocytochemistry for NKCC (T4 mouse anti-hNKCC antibody with goat anti-mouse Oregon Green 488) and for SPAK (rabbit anti-SPAK polyclonal with goat anti-rabbit Alexa Fluor 594) visualized by confocal microscopy. (A) Anti-NKCC alone (green) (B) Anti-SPAK alone (red) (C) Same field as A and B but with both channels activated (red/green); exact colocalization is yellow. NKCC and SPAK appear in the same mitochondria-rich cells, not in the apical membrane but lower in the cell (here at the plane of the nuclei) and, in most areas are exactly colocalized (yellow in C). (D) A line scan of fluorescence intensity (arbitrary units) versus distance in µm across a cell (indicated by the arrow in C) shows good correspondence in the colocalization of SPAK (red line) and NKCC (green line) in the cytosol (peaks) and not in the nucleus (central region). (E-H) As A-D except that the primary antibody for the kinase was rabbit anti-OSR1 polyclonal, not SPAK. Bars, 20 µm.