Multiplexed Fluorescence Imaging of Viral Nucleic Acids and Protein in Infected Human Lymphocytes

Abstract

Source: Shah, R., et al. Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection. J. Vis. Exp. (2020)

This video demonstrates simultaneous visualization of HIV-1 DNA, RNA, and capsid protein in infected human lymphocytes using branched DNA hybridization and immunofluorescence, enabling detailed analysis of viral replication stages within individual cells.

Protocol

1. Protein immunostaining: To label protein(s) of interest

1. Decant PBS (phosphate-buffered saline) and add 200 µL of blocking buffer (1% w/v BSA (bovine serum albumin), 10% v/v FBS (fetal bovine serum) in PBS with 0.1% v/v Tween-20 (PBST) to the coverslip. Incubate 1 h at RT (room temperature).
2. Decant blocking buffer and apply 200 µL of primary antibody diluted in PBST + 1% w/v BSA. Incubate 1 h at RT.
3. Wash the slide twice with PBST for 10 min at RT with shaking.
4. Apply a secondary antibody of choice for 1 h at RT in PBST + 1% w/v BSA.
5. Wash the slide with PBST for 10 min at RT with shaking.

2. Nuclear staining: Counter-stain nuclei following immunostaining

1. Decant PBST and apply DAPI (4',6-diamidino-2-phenylindole) or nuclear stain of choice for 1 min at RT.
2. Wash the slide twice with PBS for 10 min at RT with shaking.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-HCV NS5a antibody	Abcam	ab13833	Mouse monoclonal; works with HCV genotypes 1a, 1b, 3, and 4
Anti-HIV-1 p24 monoclonal antibody	NIH AIDS Reagent Program	3537
Anti-Mov10 antibody	Abcam	ab80613	Rabbit polyclonal
Bovine serum albumin			Blocking reagent for immunostaining
Cell media with supplements			Media appropriate for cell model
Coverslips
DAPI	ACD Bio		Nuclear stain (RNAscope kit from ACD Bio)
Dulbecco's phosphate buffered saline (1X PBS)	Gibco	14190250	No calcium and magnesium
Fetal bovine serum (FBS)			Use specific FBS based on what serum secondary antibody was raised in (e.g goat FBS)
Fisherbrand colorfrost plus microscope slides	Fisher Scientific	12-550-17/18/19	Precleaned
Slides
Tween-20			For DNA hybridization buffer and PBS-T