An Immunofluorescence Technique for Viral Protein Localization in Infected Cells

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 slide in growth medium for overnight incubation at 37 degrees Celsius, with 5% carbon dioxide.

It is important to rock the slide thoroughly to ensure an even distribution of the cells while taking care to avoid spilling the culture medium. The next day, replace the supernatants of the 70% to 80% confluent cultures with Medium-199 containing 4-10 plaque-forming units per cell, and place the slide at 37 degrees Celsius with shaking for 1 hour.

At the end of the incubation, replace the supernatants with fresh growth medium, and return the cells to the incubator for the appropriate experimental infection period. For immunofluorescence staining of the virus-infected cells, quickly wash the cells three times with PBS, followed by an 8 to 10-minute incubation in 200 microliters of 4% paraformaldehyde in PBS per well. At the end of the fixation, wash the cells three times with 200 microliters of PBS per wash and permeabilize the cells with 100 microliters of 0.2% nonionic surfactant per well for 5 to 10 minutes.

Wash the permeabilized cells three times in PBS as demonstrated, and add 200 microliters of blocking buffer to each well for a 1-hour incubation at room temperature. Next, add the appropriate concentration of the primary antibody of interest to each well for a 2-hour room-temperature incubation.

At the end of the incubation, remove any unbound primary antibody with three 10-minute washes in fresh blocking buffer, and add an appropriate secondary antibody to each well for a 1-hour incubation at room temperature, protected from light.

At the end of the incubation, wash the cells three times in a blocking buffer as demonstrated, followed by one wash in PBS, and add one drop of anti-fade mounting medium, supplemented with DAPI to each well. Then place a coverslip over the slide, and seal the coverslip with transparent nail polish.

Applying gentle pressure while mounting the coverslip will remove any excess mounting medium, helping to ensure the acquisition of high-quality images.

To analyze the nuclear and cytoplasmic distribution of ICP0, open the project in the confocal application software and select an image. Open the Quantity tab and select Sort Regions of Interest from the Tools menu. Select Draw Line, and draw a longitudinal line across the cell to be analyzed. A histogram will appear showing the fluorescence intensity along the line for both the protein of interest and DAPI.

Based on the background staining, set up a constant threshold for the intensity of the protein of interest for analysis of the subcellular distribution of the protein in each experiment. If the protein signal, on average, is below the threshold in the nuclear region, but is above the threshold beyond the DAPI boundary, categorize the protein signal as predominantly located within the cytoplasm.

If the protein signal is above the threshold throughout the nucleus and beyond the boundary of the DAPI signal, group the protein signal as a nucleus plus cytoplasmic localization. If the protein signal is above the threshold in the nucleus, but on average is below the threshold outside the boundary of the DAPI signal, group the protein signal as a nuclear localization.