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.