Terminal Transferase-Mediated dUTP Nick End-Labeling—TUNEL Assay: An In Situ Method to Detect DNA Fragmentation in Apoptotic Cells
DEŞİFRE METNİ
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.
