Indirect immunofluorescence of HEK293 cells transfected for expression of human aquaporin-4 (AQP4). A) HEK293 cells were transfected with the human aquaporin-4 (AQP4) gene and show green fluorescence at the cell membrane due to expression of green fluorescent protein (GFP)-AQP4 fusion protein at cell membranes. B) indirect immunoflurescence of these cells, using rabbit anti-human AQP4 antibody (Santa Cruz, CA) as the primary antibody and goat anti-rabbit antibody conjugated with rhobdamine, shows red fluorescence at the cell membrane due to expression of human AQP4 at cell membrane. C) overlap of A and B shows yellow fluorescence due to co-localization of GFP and AQP4 in cell membrane; and D) overlap of image C with staining for cell nuclei by DAPI clearly reveals AQP4 expression at cell membranes. E) HEK293 cells were transfected with empty vector and show green fluorescence over cell cytoplasm due to expression of GFP which, in the absence of AQP4, is not anchored in the cell membrane; and F) indirect immunofluorescence of these cells with rabbit anti-human AQP4 antibody reveals no red fluorescence, consistent with a lack of expression of AQP4.