This article describes a method for detecting apoptotic cell death in neurons using the TUNEL assay. The process involves treating brain slices with proteinase-K and fluorescent dye-labeled dUTPs to visualize DNA fragmentation.
Begin with a pre-fixed, deparaffinized, and rehydrated coronal brain slice containing neurons undergoing apoptosis, characterized by cell death and DNA fragmentation.
To detect apoptotic cell death using the TUNEL assay, treat the brain slice with the enzyme proteinase-K.
Proteinase-K enters the neurons and inactivates DNases, preventing DNA degradation.
Rinse the slice to remove any residual enzymes.
Add a buffer containing fluorescent dye-labeled deoxyuridine triphosphates, or dUTPs, and the terminal deoxynucleotidyl transferase, or TdT enzyme, then incubate.
In apoptotic neurons with abundant DNA fragments, the TdT enzyme attaches these fluorescent dUTPs to the free 3′-hydroxyl groups of the DNA fragments.
In contrast, the DNA of healthy neurons remains unlabeled.
Stain the tissue with DAPI, a nuclear dye that binds to the DNA in both healthy and apoptotic neurons, staining their nuclei blue.
Upon visualization, neurons containing both blue and green fluorescence confirm apoptotic cell death in the brain tissue.
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