IN-SITU DNA FRAGMENTATION ASSAY FOR DETECTION OF APOPTOSIS IN PARAFFIN-EMBEDDED TISSUE-SECTIONS - TECHNICAL CONSIDERATIONS

Detection, by light microscopy, of cells in situ undergoing apoptosis has been improved by use of an in situ apoptosis (DNA fragmentation) a ssay on formalin-fixed and paraffin-embedded tissue sections. We studi ed conditions of tissue preparation and fixation that may affect the t est results. In this study, we intended to determine whether archival tissues prepared under unknown conditions can be used for the in situ apoptosis assay. All tissue sections were pretreated with Proteinase K , followed by incubation with biotinylated 11-deoxyuridine triphosphat e in terminal deoxynucleotidyl transferase and then avidin-biotin-pero xidase complex. The following formalin-fixed and paraffin-embedded his tologic sections were tested: (1) normal tissues from surgically resec ted specimens fixed immediately or stored at 4 degrees C and then fixe d after 1, 2, 4, 6, or 24 hours; (2) archival autopsy material from hi stologically normal tissues; and (3) freshly prepared normal tissues f rom C57 mice. We observed that fixation- and prefixation-elapsed times do not adversely affect the results of the assay. Similar, if not ide ntical results were seen in archival human tissues stored for up to 25 years, the normal tissues freshly prepared from surgical specimens, a nd the tissues from C57 mice. We conclude that the in situ assay of DN A fragmentation is rapid, sensitive, and reproducible. The use of form alin-fixed and paraffin-embedded archival material as old as 25 years opens the way for a variety of studies of apoptosis in diverse patholo gic states.