Chapter 2 Identification of Dying Cells—In Situ Staining

Publisher Summary The basic requirements for any methodology of programmed cell death (PCD) detection include (1) resolution at the individual cell level, and (2) in situ applicability while preserving the tissue architecture. To investigate PCD in its physiological context, the chapter develops a method that satisfies both criteria that is referred to as “terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL).” This technique is based on the observation that PCD is associated with DNA degradation. Researchers have come to consider the appearance of the ladder of nucleosomal DNA on agarose gels as the hallmark of PCD. The TUNEL method relies on the in situ labeling of DNA breaks in individual nuclei in tissue sections processed through the routine procedures of histopathology. TUNEL relies on the specific binding of TdT to exposed 3′-OH ends of DNA followed by the synthesis of a labeled polydeoxynucleotide molecule. Nuclear DNA on histological sections is first exposed by proteolytic treatment; then TdT is used to incorporate biotinylated deoxyuridine into the sites of DNA breaks. The signal is amplified by avidin-peroxidase, enabling conventional histochemical identification of PCD by light microscopy.