Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

In this protocol, two antibodies from the same host can be used together in the immunofluorescence assay to study host cell and pathogen interactions. Since there are only few commercial antibodies available to recognize specific cell structures and proteins in parasites, this protocol can be very useful. This is an easy-to-perform double-labeling immunofluorescence protocol that uses polyclonal and monoclonal antibodies raised in the same species.

Many researchers may be unaware that this is possible. This approach helps when the source of antibodies is limited. It can be used to detect the pathogens in the host cell proteins in infected host cells and can also be applied to free living organisms.

This is a simple protocol, require one blocking step between the first and second pair of antibodies. The procedure will be demonstrated by Dr.Camila Gachet-Castro and PhD student Lays Trajano-Silva from my laboratory. Three days after infecting LLC-MK2 cells with Trypanosoma cruzi, collect the supernatant from the infected cells in a 15 milliliter cell culture conical tube.

Centrifuge the sample to lower cell debris, then place the tube at room temperature to allow the trypomastigotes to swim to the supernatant. After 10 minutes, collect the supernatant in a new conical tube, then centrifuge the sample and discard the supernatant before resuspending the pellet containing the parasites in complete RPMI medium. Add LLC-MK2 cells in 24-well plates containing UV sterilized rounded coverslips and allow the&m 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 five times with PBS, then fix the cells with 2%paraformaldehyde in PBS. After 10 minutes, wash the coverslips three times for five minutes each with PBS and then permeabilize the coverslips with non-ionic detergent for 10 minutes.

After three five-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 PBS 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 in blocking solution as demonstrated previously, incubate them in the mouse polyclonal antibody for 30 minutes. Next, wash the coverslips three times for five 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 IgG 2B antibody and phalloidin.

Then following three PBS washes, apply anti-fade mounting reagent as demonstrated previously. For triple labeling, after blocking the coverslips and incubating with the mouse polyclonal antibody, followed by goat anti-mouse antibody, start a new incubation with rabbit polyclonal antibody in blocking solution for 30 minutes. Then after three PBS washes, incubate the coverslips with goat anti-rabbit antibody for 30 minutes.

Next, following three PBS washes, perform a blocking step with AffiniPure rabbit anti-mouse antibody diluted in blocking solution for 30 minutes. Then wash the coverslips again before incubating with any mouse monoclonal IgG subclass antibody for 30 minutes. Following three PBS washes, incubate the coverslips for 30 minutes with a specific pair of goat anti-mouse IgG subclass antibodies.

After incubation, wash the coverslips three times for five minutes each with PBS. Analyze the immunofluorescent samples using a confocal microscope with a 63X oil immersion objective and detect the fluorescence with a photomultiplier tube and hybrid detector. Then acquire all confocal images with separated channels and perform image processing using Adobe Photoshop.

These confocal microscopy images show results with the control experiments of infected and non-infected cells highlighting the specificity of the antibodies in the host cell and the internalized parasite. The mouse polyclonal antibody anti-Trypanosoma cruzi FAZ recognized the T.cruzi giant protein in the flagellum attachment zone at the parasite flagellum, but not in the host cell. Distribution of the heterogeneous nuclear ribonucleoprotein A1 in the nuclei was observed using a commercial mouse monoclonal antibody that recognizes only the host mammalian cells, but not the parasite.

Also, the host and parasite nuclei and the parasite kinetoplasts were stained with DAPI and host F-actin was stained with phalloidin conjugated to Alexa 594, confirming the specificity of the antibodies. Double-labeling immunofluorescence shows the protein distributions in the host and the parasite analyzed by confocal microscopy. The labeling suggested no cross-reaction between antibodies using this methodology.

In summary, the protocol described here to study host-pathogen interactions presents a basic and elaborate technique that can be adapted to any combination of antibodies. This approach makes immunofluorescence cost effective and can help study the localization of two or more proteins of interest when few antibodies are available.