A Double-Labeling Immunofluorescence Technique to Visualize Host and Pathogen Proteins in an Infected Cell

Add LLC-MK2 cells in 24-well plates containing UV-sterilized rounded coverslips and allow them to settle for 16 hours. Then to infect the cells, add supernatant containing T. cruzi to each well, and incubate for six hours. Next, wash the coverslips containing the infected and non-infected cells 5 times with PBS.

Then, fix the cells with 2% paraformaldehyde and PBS. After 10 minutes, wash the coverslips three times for 5 minutes each with PBS, and then permeabilize the coverslips with non-ionic detergent for 10 minutes.

After three 5-minute PBS washes, incubate the coverslips in blocking solution for 30 minutes, followed by another 30-minute incubation with either a mouse monoclonal or polyclonal antibody. After washes, incubate the coverslips for 30 minutes with secondary antibody in blocking solution and phalloidin to stain actin filaments in the host cell.

Then, wash the coverslips with PBS three times again before applying a small amount of anti-fade mounting reagent with DAPI medium to the surface of the slide. Using forceps, gently tilt the coverslip in the medium to prevent bubble formation.

For double labeling, after incubating the coverslips and blocking solution as demonstrated previously, incubate them in the mouse polyclonal antibody for 30 minutes. Next, wash the coverslips three times for 5 minutes each with PBS before incubating them with the secondary antibody for 30 minutes.

Then following three PBS washes, perform a second blocking step with AffiniPure rabbit anti-mouse IgG diluted in blocking solution. After 30 minutes, wash the coverslips with PBS again before incubating them with the mouse monoclonal antibody for another 30 minutes.

Next, after three PBS washes, incubate the coverslips with goat anti-mouse IgG2b antibody and phalloidin. Then, following three PBS washes, apply anti-fade mounting reagent as demonstrated previously.