Immunofluorescence Assay to Detect Hepatitis C Virus Replication Complexes in Infected Cells

Take Hepatitis C virus RNA or HCV RNA-transfected hepatoma cells. The RNA encodes an HCV protein that helps replicate viral single-stranded RNA into double-stranded RNA or dsRNA.
Add a fixative to permeabilize the cellular membranes and preserve cellular integrity.
Remove the fixative and wash with a buffer to ensure complete fixative removal.
Add a blocking solution to prevent non-specific antibody binding.
Add two primary antibodies, one specific to the HCV protein, and the other targeting viral dsRNA.
Incubate to facilitate the binding of the primary antibodies to the HCV protein and dsRNA.
Wash to remove unbound antibodies.
Introduce fluorophore-conjugated secondary antibodies and incubate with agitation.
The secondary antibodies bind to their specific primary antibodies.
Wash to remove unbound secondary antibodies.
Add a nuclear dye to stain the cell nuclei.
Observe under a fluorescence microscope.
Colocalization of dual signals confirms replication of HCV within the host cell.
Fix the HCV RNA-transfected cells using methanol for 30 minutes at negative 20 degrees Celsius for the immunofluorescence assay.
When finished, wash the cells with PBS 3 times. After blocking with immunofluorescence assay blocking buffer, use rabbit polyclonal anti-NS5A primary antibody and mouse monoclonal anti-dsRNA antibody J2 at a dilution of 1 to 200, and incubate for five hours to overnight in a four degrees Celsius cold room.
Following incubation, wash the cells with PBS 3 times after the primary antibody. Then, add goat anti-rabbit IgG-488 polyclonal secondary antibody and goat anti-mouse IgG-594 polyclonal secondary antibody at a one-in-1000 dilution and incubate for one hour at room temperature on a tabletop rocker.
After washing the cells with PBS 3 times, stain the nuclei using Hoechst dye and view using a fluorescent microscope.