Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2

Abstract

Source: Kula-Pacurar, A., et al. Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization. J. Vis. Exp. (166), (2020).

This video demonstrates the application of Hybridization Chain Reaction (HCR)-Fluorescence In Situ Hybridization (FISH) for visualizing the spatial distribution of SARS-CoV-2 subgenomic RNA. Combining HCR with immunofluorescence techniques has the potential to provide insights into host-virus interactions at the single-cell level.

Protocol

1. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips

DAY 1

1. Fixing and permeabilizing cells​
   1. Fix infected cells with 3.7% w/v PFA solution for 1 h at RT.
   2. Aspirate the 3.7% PFA solution, and wash the cells twice using 1x PBS.
   3. Permeabilize the cells with PBST solution for 10 min at RT with agitation.
   4. Aspirate the PBST, and wash the cells twice with 1x PBS.
2. Detection
   1. Aspirate the 1x PBS solution, and wash the cells twice with 2x SSC at RT.
   2. Aspirate the 2x SSC so....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Confocal Microscope LSM 880	ZEISS
Incubator Galaxy170R	New Brunswick	CO170R-230-1000
Materials
15 mm x 15 mm NO. 1 coverslips	LabSolute	7695022
1.5 mL tubes	FL-MEDICAL	5.350.023.053
12-well plate	TTP	92412
Alexa fluorophore 488-conjugated secondary antibodies	Invitrogen
β5-tubulin	Santa Cruz Biotechnology	sc-134234
DAPI	Thermo Scientific	D1306
HCR Amplification Buffer	Molecular Instruments, Inc	BAM01522	Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HCR amplifier B1-h1 Alexa Fluor 647	Molecular Instruments, Inc.	S013922
HCR amplifier B1-h2 Alexa Fluor 647	Molecular Instruments, Inc.	S012522
HCR Probe Hybridization Buffer	Molecular Instruments, Inc.	BPH03821	Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HCR probe set for SARS-CoV-2 Ncapsid	Molecular Instruments, Inc.	PRE134
HCR Probe Wash Buffer	Molecular Instruments, Inc.	BPW01522	Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
Fluorescence Spectraviewer			Modeling spectral parameters
ImageJ Fiji			Acquiring and processing z-stack images
ImmunoFluorescence (IF)	Blocking	(1% BSA in PBST) 30 min at 37 °C
	Primary antibody incubation	(Antibody solution of apropriate concentration in blocking solution) 2 h at room temperature / overnight at 4 °C, 30-50 µL	(Antibody solution of apropriate concentration in blocking solution) 2 h at room temperature / overnight at 4 °C, 100 µL
	Primary antibody washing	(PBST) 3 x 5 min at room temperature
	Secondary antibody incubation	(Antibody solution of apropriate concentration in blocking solution) 1 h at 37 °C, 30-50 µL	(Antibody solution of appropriate concentration in blocking solution) 1 h at 37 °C, 100 µL
	Secondary antibody washing	(PBST) 3 x 5 min at room temperature
	Nuclear staining	(DAPI solution) 10 min at room temperature, then 2 x 5 min with 1x PBS