Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labelling techniques.
Recently developed techniques based on 'in situ end-labelling' (ISEL) of DNA strand breaks may help to identify apoptotic cells in tissue sections. We have applied ISEL techniques at the electron microscopic (EM) level, in order to verify if ultrastructural features of apoptosis are indeed associated with evidence of DNA fragmentation, and whether cells committed to, but which have not yet entered the stage of cell death are also labelled. Terminal transferase and DNA polymerase assays were applied to thin sections of Araldite and LR Gold-embedded medulloblastomas and embryonic mouse dorsal root ganglia. Digoxigenin-labelled nucleotides were used; incorporation was demonstrated by immunogold staining. Apoptotic cells in various stages of the death process were easily labelled in both tissues. In addition, DNA fragmentation was demonstrated in cells with initial chromatin condensation, but otherwise indistinguishable from adjacent unstained cells. Our results show that EM-ISEL techniques effectively demonstrate the occurrence of DNA strand breaks in apoptotic and possibly 'pre-apoptotic' cells in neural tissues. Since the labelling is easily obtained on tissue that is routinely processed for electron microscopy, this technique may allow retrospective studies on archival material.
Migheli
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Clinica Neurologica II, Università di Torino, Italy.
Attanasio
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Schiffer
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D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002528:Cerebellar Neoplasms / Q000235:genetics / Q000648:ultrastructure*; D004249:DNA Damage; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I; D004273:DNA, Neoplasm / Q000235:genetics; D005727:Ganglia, Spinal / Q000196:embryology / Q000648:ultrastructure*; D006801:Humans; D008527:Medulloblastoma / Q000235:genetics / Q000648:ultrastructure*; D051379:Mice; D008854:Microscopy, Electron; D016877:Oxidants; D013194:Staining and Labeling / Q000379:methods; D016610:Tissue Embedding / Q000379:methods
D004273:DNA, Neoplasm; D016877:Oxidants; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I