*2.6. Immunocytochemical and Immunoblot Detection of Viral Proteins*

Expression of HCV proteins in the transfected MSCs was determined by the methods of indirect immunofluorescence and immunoperoxidase staining, using monoclonal antibodies (mAbs) against HCV proteins [33] as primary antibodies and secondary antibodies againstmouseimmunoglobulins (Ig) conjugated with fluorescein isothiocyanate (FITC) or horseradish peroxidase (HRP) (Dako, Denmark), as previously described [34,35]. Cell nuclei were stained with 4- -6-diamidino-2-phenylindole (DAPI) (immunofluorescence analysis) or with hematoxylin (immunoperoxidase method). The signals were visualized using an Axio Scope A1 microscope (Zeiss, Germany). The proportion of cells expressing viral proteins relative to the total number of cells was counted in at least eight fields of view at a magnification of 400× and expressed as a percentage value. This corresponds to counting at least 1600 cells for each HCV protein.

Western blot analysis was performed as described previously using the same monoclonal antibodies or serum of the rabbits immunized with the respective protein [36].
