Immunostaining to Visualize Viral Antigen and Host Protein Colocalization in the Insect Gut

Begin with a chemically fixed gut excised from an insect carrying a plant virus.
Wash with a buffer to remove the fixative.
Add a non-ionic detergent to permeabilize the cellular membranes.
Wash again to remove any detergent residues.
Add a solution containing two different fluorescent antibodies and a blocking agent.
Upon incubation, the blocking agent saturates nonspecific binding sites, while the antibodies enter the cytoplasm.
One antibody binds to the viral antigen within the host cell vesicles, and the other binds to a vesicle-associated membrane protein.
Wash to remove the unbound antibodies.
Add a fluorescent dye that binds to the actin filaments to visualize the host cells.
Wash to eliminate any unbound dye.
Mount the stained gut in a medium containing a fluorescent nuclear stain to label the cellular DNA.
Using confocal microscopy, visualize the coexistence of the green and red fluorescent signals, indicating colocalization of the viral antigen and the membrane protein.

Prepare the antibodies and mounting medium as described in the text manuscript.

Place the freshly excised and PBS washed WBPH guts in 100 microliters of 4% paraformaldehyde in a 200 microliter centrifuge tube at room t