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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.
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