Therefore, TdT labeling tends to be stronger than that with DNA polymerase. In addition, some tissue samples (breast cancer) that are TUNEL-positive do not even label with the ISNT method leading to the hypothesis that DNA breaks in this case of apoptosis probably do not produce 3′
recessed OH groups.46 The in situ labeling method described in this chapter will be restricted to the TUNEL method.
The labeling technique in the TUNEL method is functionally based on the activity of the TdT enzyme that transfers dUTP to the 3′-OH groups of the fragmented DNA.14 The dUTP moiety is chemically tagged, thus allowing the visualization of cells that are apoptotic. Normal nuclei have very low
levels of 3′-OH breaks and do not produce any appreciable signal as opposed to apoptotic nuclei. In the original method, dUTP was labeled with biotin, and detection was achieved with avidin-peroxidase followed by color development. 14