A Double-Labeling Immunofluorescence Technique to Visualize Host and Pathogen Proteins in an Infected Cell

Begin with fixed and permeabilized epithelial cells infected with a pathogenic parasite.

Add a blocking buffer to prevent non-specific antibody binding.

Overlay with mouse polyclonal primary antibodies, specifically interacting with the parasite's proteins.

Add far-red fluorophore-labeled secondary antibodies that interact with parasitic protein-bound primary antibodies.

Wash to remove unbound antibodies. Block with anti-mouse antibodies that bind to primary antibodies, minimizing the cross-reactivity during host-protein labeling.

Add mouse monoclonal primary antibodies targeting host ribonucleoproteins within epithelial cells.

Overlay with a mix of green fluorophore-labeled secondary antibodies and red fluorophore-conjugated phalloidin, facilitating the labeling of host protein-bound primary antibodies and actin, respectively.

Wash with a buffer. Mount using a DNA stain-containing medium, imparting a blue coloration to the host nucleus, parasite nucleus, and kinetoplast.

Analyze the samples using a confocal microscope. The double-labeling immunofluorescence technique shows green host proteins and red parasitic proteins, enabling their precise subcellular localization.