Aug 04, 2025
DENGUE Antiviral Immunofluorescence staining Protocol
- 1Icahn School of Medicine at Mount Sinai;
- 2ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Briana McGovern 2025. DENGUE Antiviral Immunofluorescence staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv52yb6v1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2025
Last Modified: August 04, 2025
Protocol Integer ID: 123553
Keywords: SARS-CoV-2, immunofluorescence, antiviral staining, Alexa Fluor 488, cell fixation, permeabilization, fluorescence microscopy, dengue antiviral immunofluorescence, dengue, ic7c7 primary antibody, infected cells for quantification, conjugated secondary antibody, secondary antibody, primary antibody, immunofluorescence, viral infection, labeled infected cell, using ic7c7, staining protocol, fixed sar
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This protocol describes immunofluorescence staining of fixed SARS-CoV-2 infected cells using IC7C7 primary antibody (produced in house against SARS-CoV-2 NP) and Alexa Fluor 488-conjugated secondary antibody. The procedure results in fluorescently labeled infected cells for quantification of viral infection.
Protocol materials
0.1% Triton X-100-containing 1XPBS solution
Tween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS
Goat anti-Rabbit IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Thermo Fisher ScientificCatalog #A-11008
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Assay is performed under biosafety level 3 conditions.
Post-fix prep
Once you have the cells fixed with 10% PFA for minimum 24 hours you may handle the plates.
Remove PFA from the wells and discard in the fume hood.
Wash the wells 3x with 100ul PBS.
Blocking
Add 100ul 1% PBS-BSA to the wells
Incubate for 1 hour at RT, then remove
Permeabilization
Treat the wells with 100ul 0.1% Triton X-100-containing 1XPBS solution for 10-15 minutes.
Remove the triton, and wash 3x with 100ul 0.1% Tween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS
Primary Antibody
Treat the wells with 50 ul primary flavivirus envelope protein antibody 4G2 (generated in house by [email protected]) at 0.001mg/ml (1:1000) in 1% BSA
Incubate overnight at 4C. If urgent, incubate for 1-2 hours at RT
After incubation, wash 2x with 100ul 0.1% Tween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS
Secondary Antibody
Treat the wells with 50ul secondary antibody Goat anti-Rabbit IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Thermo Fisher ScientificCatalog #A-11008 (1:1000) and DAPI in 1% BSA
Incubate overnight at 4C. If urgent, incubate 1-2 hours at RT
After incubation, wash 2x with 100ul 0.1% Tween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS
Add 100ul plain PBS to the wells for reading and storage