This article describes a method for detecting apoptosis in microbial pathogen-infected host cells using the TUNEL assay. The process involves treating tissue samples to prevent DNA degradation and visualizing apoptotic cells through fluorescence microscopy.
Microbial pathogen-infected host cells undergo apoptosis - programmed cell death. During apoptosis, DNases degrade the chromosomal DNA into small fragments, leading to cell death.
To detect apoptosis using the TUNEL assay, take a glass slide with a deparaffinized and rehydrated skin tissue section infected with a fungal pathogen.
Treat the tissue with proteinase-K to inactivate the cellular DNases, preventing them from potentially degrading the DNA during analysis. Add hydrogen peroxide to quench the reaction, and wash the slide to remove unreacted proteinase-K.
Add a buffer containing digoxigenin-labeled deoxy-uridine triphosphates, or dUTPs. Supplement it with a terminal deoxynucleotidyl transferase, TdT enzyme, and incubate. In apoptotic cells, the TdT enzyme catalyzes the attachment of labeled dUTPs to the exposed 3′-hydroxyl groups of the fragmented DNA.
Next, add an anti-digoxigenin antibody conjugated to a rhodamine reporter, and incubate it in the dark to facilitate antibody binding to digoxigenin-labeled dUTPs, imparting red fluorescence upon imaging.
Treat the tissue samples with a fluorescent nuclear counterstain to visualize the nuclei. Place a coverslip over the tissue section, and visualize it under a fluorescence microscope.
The blue fluorescence helps identify the nuclei of all the cells in the tissue section, while the presence of red fluorescence in the rhodamine-stained cells indicates DNA fragmentation due to apoptosis.
To begin this procedure, prepare hydrophilic glass slides with sample tissue, as outlined in the text protocol.
To deparaffinize the tissue sections, wash the slides three times with xylene, for 5 minutes each, in a coplin jar. Next, wash the slides with 100% ethanol two times for 5 minutes each. After this, wash the slides with 95% ethanol, and 70% ethanol, for 3 minutes each, then, wash the slides once with PBS for 5 minutes.
To prepare experimental and negative control slides, pre-treat the tissue by adding freshly diluted Proteinase K directly to the slides. Incubate the slides for 10 minutes at room temperature, then, wash the slides in PBS two times for 2 minutes each.
To prepare positive control slides, pre-treat the tissue with DN buffer, then, incubate the slides at room temperature for 5 minutes. After this, dissolved DNase I in DN buffer to a final concentration of 0.1 micrograms per milliliter. Add the prepared DNA solution to the slides. Incubate the slides for 15 minutes at room temperature, then, wash the sides with distilled water in a coplin jar five times, for 3 minutes each.
Quench the reaction in 3% hydrogen peroxide in PBS for 5 minutes at room temperature. Next, wash the slides in PBS two times for two minutes each. Then, add equilibrium buffer to the tissue on the slides. Incubate for 10 seconds at room temperature, then, tap off the excess liquid around the tissue section.
Dilute terminal deoxynucleotidyl transferase enzyme in reaction buffer, then, add it to the tissue on the experimental and positive control slides. Incubate in a humidified chamber for 1 hour at 37 degrees Celsius.
After this, place the slides in a coplin jar containing the working concentration of stop buffer. Agitate for 15 seconds, and then, incubate the slides at room temperature for 10 minutes. Then, wash the slides with PBS three times for 1 minute each, and tap off the excess liquid.
Next, add the anti-digoxigenin conjugate pre-warmed to room temperature directly onto the tissue. Incubate the slides for 30 minutes at room temperature in a humidified dark chamber.
After the incubation is complete, wash the slides with PBS four times for two minutes each. Tap off any excess liquid. Then, add mounting medium to the sides. Add 15 microliters of DAPI solution to the mounting medium on the slides. Cover each slide with a coverslip, and seal with nail polish. Allow the slides to dry in the dark.
Using a fluorescent microscope, observe the slides immediately upon completion of the staining process.
Take photos at 400 times at random intervals along each skin section. Count DAPI-stained blue cells to ensure at least 100 cells per skin section, per animal, are visible. Next, count the number of TUNEL-positive cells which appear as red single cells with clear cell edges.
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