Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.

TLDR

Programmed cell death is essential for development and tissue homeostasis, yet its presence is usually inferred indirectly from DNA fragmentation detected by gel electrophoresis of pooled extracts. The authors sought to create a method that visualizes PCD in situ at the single‑cell level while preserving tissue architecture. They prepared protease‑treated sections, performed nick‑end labeling with biotinylated poly dU via terminal deoxy‑transferase, stained with avidin‑conjugated peroxidase, and screened multiple tissues including intestine, epidermis, lymphoid organs, ovary, and others. The assay specifically stains nuclei undergoing PCD, shows that the process starts at the nuclear periphery, lasts 1–3 h, occurs in clusters, and reveals a greater extent of tissue‑PCD than morphology‑based apoptosis, opening avenues for further research.

Abstract

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.