Multiplexed Fluorescence Imaging of Viral Nucleic Acids and Protein in Infected Human Lymphocytes

Begin with human lymphocytes infected with HIV-1 on a coverslip mounted on a slide.

These cells have been hybridized with probes that fluorescently label viral RNA and proviral DNA.

Incubate the cells in a blocking solution to prevent non-specific antibody binding.

Introduce primary antibodies targeting the HIV-1 capsid protein.

Incubate to allow the primary antibodies to bind to the capsid protein.

Wash the cells with buffer to remove unbound antibodies.

Add fluorescently labeled secondary antibodies that bind to the primary antibodies.

Wash off the unbound antibodies.

Add a DNA-specific dye to stain the nuclei of the cells, then wash to remove excess dye.

Use multiplexed fluorescence imaging to simultaneously detect viral DNA, RNA, protein, and host nuclei in distinct colors.

This single-cell visualization enables the study of different stages of the viral life cycle through simultaneous visualization of viral DNA, RNA, and proteins in infected cells.

For immunostaining of the proteins of interest, after the last wash, treat each coverslip with 200 microliters of blocking buffer for a one hour incubation at room temperature.

At the end of the incubation, decant the blocking buffer and add 200 microliters of the primary antibody of interest diluted in PBS plus Tween 20 supplemented with 1% bovine serum albumin. After a one-hour incubation at room temperature, wash the slides with two 10-minute washes in fresh PBS plus Tween 20 with shaking per wash before labeling the samples with an appropriate secondary antibody for one hour at room temperature. At the end of the incubation, wash the slides two times with fresh PBS plus Tween 20 for 10 minutes with shaking per wash.

After the second wash, counterstain the nuclei with an appropriate nuclear dye for one minute at room temperature, followed by two washes in PBS for 10 minutes at room temperature with shaking.