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

الملخص

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.

البروتوكول

1. Immunofluorescence Assay (IFA)

1. Fix the Hepatitis C virus (HCV)-infected and transfected cells using methanol for 30 min at -20 ºC for the immunofluorescence assay.

2. Wash the cell with phosphate-buffered saline (PBS) three times and block with IFA blocking buffer.

3. 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.

4. Wash the cells with PBS three times after the primary antibody.

5. 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.

6. Wash cells with PBS three times and stain nuclei using Hoechst dye and view using a fluorescence microscope (Figure 1D).

النتائج

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...

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
Dulbecco’s modified Eagle’s medium (DMEM)	Fisher Scientific	10-017-CV
Non essential amino acid	Fisher Scientific	MT25025CI
HEPES	Life Technologies	15630080
Glutamax	Life Technologies	35050061
Opti-MEM Reduced Serum Medium,no Phenol Red	Life Technologies	11058-021
Huh-7.5.1	The Scripps Research Institute		The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center
mouse monoclonal anti-dsRNA antibody J2	English & Scientific Consulting Kft.	10010200
Goat anti-rabbit IgG Alexa Fluor 488	Life Technologies	A11008
Goat anti-rabbit IgG Alexa Fluor 594	Life Technologies	A11020
Blotting Grade Blocker Non Fat Dry Milk	Bio-Rad	170-6404XTU
Tween-20	Bio-Rad	170-6531XTU
Anti-Hepatitis C Virus NS3 antibody [8 G-2]	Abcam	ab65407
Anti-Hepatitis C Virus NS3 antibody [H23]	Abcam	ab13830