Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
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.
Gavrieli
Y
Y
Department of Experimental Medicine, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Sherman
Y
Y
Ben-Sasson
S A
SA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D007082:Ileum / Q000166:cytology; D007420:Intestine, Large / Q000166:cytology; D007700:Kinetics; D008297:Male; D009928:Organ Specificity; D006080:Ovarian Follicle / Q000166:cytology; D051381:Rats; D011919:Rats, Inbred Strains; D012867:Skin / Q000166:cytology; D013950:Thymus Gland / Q000166:cytology* / Q000648:ultrastructure
D003907:Dexamethasone; D004247:DNA