Cell-based IIFA to detect AQP4 autoantibodies using transfected HEK293 cells that express human AQP4. A-C, HEK293 cells transfected with human AQP4 gene reveal green fluorescence on cell membranes due to expression of GFP-AQP4 fusion protein as a membrane protein (A). IIFA of serum from a healthy subject (negative control), using secondary goat anti-human IgG conjugated with rhobdamine, shows no red fluorescence (B) signifying seronegativity for AQP4 autoantibodies. With B overlapped with A, no yellow fluorescence is observed (C). D-F, IIFA of transfected cells expressing GFP-AQP4 fusion protein (D) with rabbit anti-human AQP4 antibody as primary antibody (positive control) reveals red fluorescence at the cell membrane (E). When E is overlapped with D, yellow fluorescence is observed (F) signifying colocalization of AQP4 with GFP at cell membranes. G-I, IIFA using transfected HEK293 cells expressing GFP-AQP4 fusion protein (G) of serum from a NMO patient seropositive for NMO-IgG shows red fluorescence at the cell membranes (H). When H is overlapped with G, yellow fluorescence due to colocalization of GFP with AQP4 is observed at cell membranes (I) signifying seropositivity for AQP4 autoantibodies. J-L, IIFA of serum of the same NMO patient in G-I using HEK293 cells transfected with empty vector without human AQP4 gene. The transfected HEK293 cells show green fluorescence in their cytoplasm due to expression of GFP, but in the absence of GFP-AQP4 fusion protein, GFP is not expressed as a membrane protein (J); and IIFA of the patient's serum, seropositive for NMO-IgG, reveals no red fluorescence at cell membranes (K) and no yellow fluorescence when overlapped with J (L). This proves that the human IgG bound to transfected HEK293 cell membrane in H and I represents autoantibodies targeting human AQP4.