Confocal Microscopy of Fluorescently Labeled EHDV-Infected Cell Cultures
Joanna Wells1, Amanda Corla1, Philippa C Hawes2,3
1The Pirbright Institute, Woking, UK.
Insights
Confocal laser scanning microscopy and immunofluorescence (IF) visualize intracellular structures. This study details an IF protocol to locate EHDV proteins in infected cells, highlighting IF
Area of Science:
- Cell Biology
- Microscopy
- Immunology
Background:
- Confocal laser scanning microscopy offers superior intracellular structure visualization compared to widefield fluorescence microscopy.
- Immunofluorescence (IF) leverages antibody specificity for protein localization within cells.
- Antibodies tagged with fluorophores enable fluorescent imaging of target proteins.
Purpose of the Study:
- To detail a robust and adaptable immunofluorescence protocol for protein localization.
- To apply this established IF protocol for identifying EHDV (Epizootic Hemorrhagic Disease Virus) proteins in infected cell cultures.
Main Methods:
- Utilized confocal laser scanning microscopy for high-resolution imaging.
- Employed immunofluorescence (IF) techniques with specific antibody-fluorophore conjugates.
- Applied a long-established laboratory protocol for IF staining and imaging.
Main Results:
- Successfully visualized and localized EHDV proteins within infected cultured cells.
- Demonstrated the utility of the IF technique for studying viral protein distribution.
Conclusions:
- Immunofluorescence is a valuable, commonly used technique for protein localization, despite not providing ultrastructural detail.
- The described IF protocol is effective for locating specific viral proteins, such as EHDV proteins, in infected cells.
Abstract:
The confocal laser scanning microscope allows the visualization of intracellular structures in greater detail than a widefield fluorescence microscope. Immunofluorescence (IF) techniques make use of the inherent ability of antibodies to bind to specific epitopes of specific proteins. Tagging these antibodies with an easily visualized molecule, e.g., a fluorophore, enables imaging in the fluorescence microscope. This is, however, a localization technique and will only give information about where certain proteins are; it does not provide the ultrastructural context provided by the transmission electron microscope. It also relies heavily on the accuracy and binding affinity of individual primary antibodies. Despite this, it is a commonly used, robust, and adaptable technique. In this chapter, we use a long-established IF protocol from our laboratory to locate EHDV proteins in a monolayer of infected cultured cells.
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