Pitfalls and Caveats in Histochemically Demonstrating Apoptosis

Abstract

For investigating the biological significance of apoptosis, the exact and sensitive histochemical identification of apoptosis and apoptotic cells is essential. However, we need to recognize both the pitfalls and caveats in performing histochemical staining and in interpreting the findings obtained. DNA fragmentation-based approaches, such as TdT-mediated dUTP-biotin nick end-labeling (TUNEL), in situ nick translation (ISNT) and immunostaining for single-stranded DNA, represent DNA alterations in the apoptotic cell, but they are technically unstable and occasionally give false positive and false negative findings. In contrast, immunostaining for intracellular proteins cleaved and activated by caspases, including cleaved caspase 3, cleaved poly(ADP-ribose) polymerase, cleaved cytokeratin 18 and cleaved actin (fractin), is technically reproducible, but the intracellular accumulation of the activated proteins is not necessarily synchronized. The present review focuses on the pretreatments for enhancing the sensitivity of these techniques, as well as their limitations and comparisons in histochemically demonstrating apoptosis and apoptotic cells.