ULTRASTRUCTURAL IDENTIFICATION OF APOPTOTIC NUCLEI USING THE TUNEL TECHNIQUE

We describe an ultrastructural adaptation of the method of terminal de oxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TU NEL) for the identification of DNA fragmentation. Thin sections of tis sue embedded in hydrophilic resin were nick end labelled with biotinyl ated dUTP which was subsequently labelled with avidin conjugated to go ld particles. The technique was validated by labelling the nuclei of L 929-8 cells treated with tumour necrosis factor alpha. These cells are known to respond to treatment with the factor by undergoing apoptosis . The method was then used on tissue from the chick embryo which is kn own to be undergoing programmed cell death. This tissue was from the n eural tube and the posterior necrotic zone of the limb bud, where cell s can be identified as undergoing apoptosis based on the morphology of their nuclei. The method specifically labelled heterochromatin adjace nt to the nuclear envelope as well as that associated with the nucleol us of cells from regions of the embryo where programmed cell death was expected. In addition to labelling the nuclei of cells that were clea rly undergoing apoptosis, the method also identified nuclei of apparen tly normal cells. This method, used in conjunction with corroborating techniques, provides a means for the early detection of cells undergoi ng DNA fragmentation, before the onset of gross apoptotic morphology, and in cells that do not show classical apoptotic characteristics.