Ej. Sanders et Ma. Wride, ULTRASTRUCTURAL IDENTIFICATION OF APOPTOTIC NUCLEI USING THE TUNEL TECHNIQUE, Histochemical Journal, 28(4), 1996, pp. 275-281
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.