A fluorescent double‐labeling method to detect and confirm apoptotic nuclei in parkinson's disease

Abstract

In situ end‐labeling (ISEL) has become a widely used method to determine whether cells die via apoptosis by detecting double‐stranded DNA breaks that are the result of endonuclease digestion. The enzyme terminal deoxynucleotidyl transferase can be used to label the digested 3′‐OH ends of DNA with biotin‐, digoxigenin‐, or fluorescent probe‐conjugated nucleotides. However, both single‐stranded and double‐stranded DNA breaks can be labeled by this method and therefore ISEL cannot unequivocally demonstrate apoptosis when used alone. We have developed a fluorescent double‐labeling method using ISEL combined with the cyanine dye YOYO‐1 that binds to DNA. When combined with confocal laser microscopy and deconvolution analysis, YOYO‐1 can demonstrate the presence or absence of nuclear chromatin condensation and thus confirm that ISEL‐positive nuclei are indeed apoptotic. Preliminary findings indicate that dopaminergic neurons in the substantia nigra compacta die via apoptosis in Parkinson's disease.