An Immunofluorescence Technique for Viral Protein Localization in Infected Cells

1. Cell Seeding and Virus Infection

1. At 20–24 h before the virus infection, seed 5 x 10⁴ of human embryonic lung (HEL) fibroblast cells or other cells to be examined on a 4-well 11 mm staggered slide in growth medium (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)). Incubate the cells at 37 °C with 5% carbon dioxide (CO₂).
   NOTE: Each well should have 70-80% cell confluency at the time of infection.
2. On the next day, remove the growth medium and infect the cells with viruses in Medium-199 at a range of 4–10 pfu/cell. Incubate virus-infected cells for 1 h at 37 °C. Keep shaking the slide during the incubation period.
3. After the 1 h incubation, remove Medium-199 and supplement with growth medium.
   NOTE: Drugs that interfere with different infection phases can be added at this step or prior to viral absorption.
4. Incubate the virus-infected cells at 37 °C with 5% CO₂ for various lengths of infection period.

2. Fixation and Permeabilization

1. At proper infection time, quickly wash the infected cells with phosphate-buffered saline (PBS) 3 times and add 200 μL of 4% paraformaldehyde freshly prepared in PBS. Incubate the cells with paraformaldehyde for 8–10 min at room temperature to fix the cells in each well.
2. Aspirate paraformaldehyde and wash the wells with 200 μL of PBS for 3 times. Completely aspirate PBS after the 3^rd wash.
3. Add 100 μL of 0.2% non-ionic surfactant to each well to permeabilize the cells for 5–10 min.
4. Aspirate the non-ionic surfactant and wash the wells with 200 μL of PBS for 3 times.

3. Immunofluorescent Staining

1. Completely aspirate PBS and add 200 μL of blocking buffer (1% bovine serum albumin (BSA) and 5% horse serum in PBS) in each well and incubate at room temperature for 1 h or at 4 °C overnight.
2. Add experimentally determined concentration of primary antibody against infected cell protein 0 (rabbit anti-ICP0 polyclonal antibody) in blocking buffer and incubate primary antibody at room temperature for 2 h or at 4 °C overnight.
3. Wash with blocking buffer 3 times with 10 min incubation. Add Alexa 594-conjugated goat anti-rabbit secondary antibody (1:400 diluted in blocking buffer) and incubate the slides at room temperature for 1 h. Then wash the slides 3 times with blocking buffer at 10 min intervals.
4. Finally wash the slide once with PBS to remove residual BSA and horse serum.
5. Add one drop of antifade mounting medium with 4',6-diamidino-2-phenylindole (DAPI) to mount the slide and seal it with coverslip using transparent nail polish.

4. Confocal Imaging

1. With a confocal microscope, set the wavelength at 590–650 nm for Alexa 594 and 410–520 nm for DAPI. Select image format at 1024 x 1024 and line average of 8 to acquire high resolution images.
2. Analyze each well on the 4-well slide under a confocal microscope. Acquire representative cell images under the 100X objective, as shown in Figure 1 and Figure 2.
3. For counting large numbers of cells, take images of consecutive fields under the 40X objective.
   NOTE: It requires 5–10 images to accumulate over 200 infected cells from each time point of each infection.
4. In each experiment, take pictures with constant confocal parameters for all samples that need to be compared.

5. Analyzing Nuclear vs. Cytoplasmic Distribution

1. Open project with the confocal application software. Select an image from which cells need to be tabulated for nuclear vs. cytoplasmic distribution of ICP0.
2. Click the tab "Quantity" from top menu and select "sort ROIs" from tools menu.
3. Draw a longitudinal line across the cell to be analyzed by selecting "Draw line" from top menu.    
   NOTE: Histogram will appear showing the fluorescence intensity along the line for both ICP0 and DAPI. In the histogram, blue line represents DAPI pixels and marks the boundary of the nucleus whereas the red line represents ICP0 pixels.
4. Based on background staining, set up a constant threshold for ICP0 intensity to analyze ICP0 subcellular distribution in each experiment.
   1. As exemplified in Figure 2, if the red signal on average is below the threshold in the nuclear region but is above the threshold beyond the blue boundary, categorize the red signal as predominantly located in the cytoplasm.
   2. If the red signal is above the threshold throughout the nucleus and beyond the boundary of blue signal, group the red signal as nucleus plus cytoplasmic localization.
   3. If the red signal is above the threshold in the nucleus but on average is below it outside the boundary of blue signal, group the red signal as nuclear localization.
5. Tabulate more than 200 infected cells from each sample at different infection time and plot in bar graph to illustrate ICP0 movement according to time (Figure 3).