DEMONSTRATION OF APOPTOTIC CELLS IN TISSUE-SECTIONS BY IN-SITU HYBRIDIZATION USING DIGOXIGENIN-LABELED POLY(A) OLIGONUCLEOTIDE PROBES TO DETECT THYMIDINE-RICH DNA-SEQUENCES

The recognition of apoptotic cells by morphological appearance alone m ay be difficult. We have investigated the use of in situ hybridization (ISH) with digoxigenin-labeled poly(A) probes to detect apoptotic cel ls in tissue sections. This method was compared to conventional morpho logic assessment and in situ end-labeling (ISEL) in a range of tissues in which apoptosis is known to occur. ISH with poly(A) probes detecte d apoptotic nuclei in all tissues in which there was evidence of apopt osis as judged by conventional histology. ISH and, to a lesser extent, ISEL preferentially labeled shrunken but still intact nuclei with mar gination of chromatin, presumably at an early stage of apoptosis. The poly(A) hybridization was abolished by pretreatment of tissue sections with DNAse. After denaturation of DNA, poly(A) hybridized to nuclei i n all cells. No convincing hybridization signal was detected in alcoho l-fixed or fresh-frozen sections. Both ISEL and ISH labeled some of th e nuclei in ischemic tissues. ISH with poly(A) oligonucleotide probes offers a simple alternative to ISEL for detection of cells in early st ages of apoptosis. These probes hybridize to thymidine-rich sequences of DNA in the highly repeated Alu sequences within the nuclear genome. These sequences are believed to become available for hybridization af ter formalin fixation and paraffin embedding as a result of the apopto sis-related increase in the susceptibility of nuclear DNA to denaturat ion.