Determining Viral RNA Distribution in Host Cells by Fluorescence In Situ Hybridization

Begin with a slide containing permeabilized, virus-infected host cells, where viral RNAs are distributed between the nuclear and cytoplasmic compartments.

Add a pre-hybridization buffer and incubate under humid conditions.

In the buffer, blocking reagents block nonspecific binding sites, while RNase inhibitors inactivate RNases and preserve RNA integrity.

Remove the buffer and add denatured, digoxigenin-tagged oligonucleotide probes complementary to viral RNA.

Incubate in the dark to allow probe-RNA hybridization, then wash repeatedly.

Add a fixative to stabilize the probe-RNA hybrids, then wash repeatedly. Add a detergent-supplemented buffer to maintain cell permeability.

Add a green-fluorophore-conjugated anti-digoxigenin antibody that interacts with digoxigenin, then wash repeatedly.

Fix the cells to preserve the complexes, then wash again.

Add a DNA-binding dye to stain the nuclei. Wash excess dye repeatedly.

Mount with a coverslip and seal it.

Under a confocal microscope, visualize the green fluorescence, reflecting the distribution of viral RNA between the host cell nucleus and cytoplasm.

After three PBS washes as demonstrated, fix the samples again in 4%formaldehyde in PBS for 10-15 mi