Subsequently, several modifications of the original method have been made. One such change involves the chemical modification of dUTP to digoxigenin-dUTP, which is then detected by using an antibody that recognizes the digoxigenin group. 48 This antibody can itself be fluorescently labeled or can be detected by the addition of a horseradish peroxidase (HRP) conjugated secondary antibody followed by DAB color development that results in brown apoptotic nuclei. A more recent and simple technique involves the direct tagging of dUTP with the fluorescent molecule, fluorescein.49
This modification drastically reduces the time required to label apoptotic cells. In addition, the direct fluorescent TUNEL method produces significantly lower levels of background fluorescence when compared to the method that employs fluorescently labeled antidigoxigenin antibody. In experiments where the detection is based on a color reaction, the cells are counterstained with a staining reagent such as methylene blue.