An Immunofluorescence Technique for Viral Protein Localization in Infected Cells

Begin with a slide containing adhered host cells.

Add viruses and allow them to enter the host cells.

Remove the spent medium. Add fresh medium to enable viral protein synthesis and transport.

Use paraformaldehyde to preserve cell morphology and protein location.

Add detergent molecules to make the cells permeable.

Apply a blocking buffer to block non-target binding sites.

Add primary antibodies that bind target viral proteins. Remove unbound antibodies.

Overlay with red fluorophore-conjugated secondary antibodies for fluorescently tagging the viral proteins. Remove unbound antibodies.

Add a fluorescent nuclear dye-containing mounting medium to stain the nuclei.

Using confocal microscopy, observe the location of red-fluorescent viral proteins and blue-fluorescent host nuclei.

A stronger red signal around the blue area indicates cytoplasmic localization, while a stronger signal on the inside suggests nuclear localization.

Equal signal levels in and around the blue area indicate that proteins are located in both the nucleus and cytoplasm.

20 to 24 hours before the virus infection, seed 5 x 10⁴ human embryonic lung or HEL fibroblast cells per well onto a 4-well 11-millimeter staggered