Fig. (3) Colocalization of HuR with HDV antigens and RNA. (A-C) Double indirect immunofluorescence was used to detect HDAg and HuR proteins in cultured Huh7-D12 cells. Cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100, and stained with anti-HDAg antibody (A, green staining) and anti-HuR antibody (B, red staining). In situ hybridization followed by immunofluorescence was performed to detect HDV RNA and the HuR protein. After fixation and permeabilization as above, cells were hybridized with a digoxigenin-labeled probe to detect HDV RNA (D, green staining) and with an anti-HuR antibody (E, red staining). Overlaps of images are shown in panels C and F. The JaCoP plugin and ImageJ program were used to determine the Mander´s overlap coefficient.