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

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Resumen

Source: Ren, S., et al. A Protocol for Analyzing Hepatitis C Virus Replication. J. Vis. Exp. (2014).

This video demonstrates an immunofluorescence-based method to detect Hepatitis C virus replication in infected hepatoma cells. The protocol involves cell fixation, antibody staining for viral protein and double-stranded RNA, and visualization using fluorophore-tagged secondary antibodies. Dual signal detection under a fluorescence microscope confirms active viral replication within the host cells.

Protocolo

1. Immunofluorescence Assay (IFA)

Fix the Hepatitis C virus (HCV)-infected and transfected cells using methanol for 30 min at -20 ºC for the immunofluorescence assay.
Wash the cell with phosphate-buffered saline (PBS) three times and block with IFA blocking buffer.
Use rabbit polyclonal anti-NS5A primary antibody or mouse monoclonal anti-double-stranded RNA (dsRNA) antibody J2 at a dilution of 1:200 (1 μg/ml) and incubate for 5 hr to overnight at 4 ºC.
Wash the cells with PBS three times after the primary antibody.
Add goat anti-rabbit Immunoglobulin G (IgG)-488 polyclonal secondary antibody or goat anti-mouse IgG-594 polyclonal secondary antibody at a 1:1,000 dilution (1 μg/ml) and incubate for 1 hr at room temperature.
Wash cells with PBS three times and stain nuclei using Hoechst dye and view using a fluorescence microscope (Figure 1D).

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Resultados

Figure 1. Assessing the growth phenotypes of HCV constructs. A) Evaluating the genome replication kinetics of wild-type (WT) and polymerase null (Pol-) viruses. The genome copy numbers of the sense RNA strand assessed by RT-qPCR are present...