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In Situ Detection of Programmed Cell Death in Senescing Nicotiana tabacum Leaves Using TUNEL Assay
Branka Uzelac1, Dušica Janošević2, Snežana Budimir3
1Institute for Biological Research "Siniša Stanković", University of Belgrade, Belgrade, Serbia. branka@ibiss.bg.ac.rs.
Abstract:
Leaf senescence constitutes a highly regulated final phase of leaf development, leading to cell death that is recognized as a type of programmed cell death (PCD). Degradation of nuclear DNA into oligonucleosomal fragments (DNA ladder) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay are methods commonly used to detect PCD-specific DNA cleavage. TUNEL reaction in situ labels free 3'-OH DNA strand breaks (nicks), thus allowing histological localization of nuclear DNA degradation during PCD. Here we describe in situ labeling of PCD-specific nuclear DNA fragmentation on conventional histological sections of senescing tobacco leaves. Incorporation of fluorescein-labeled dUTPs is detected by fluorescence microscopy, which enables in situ visualization of PCD at the single-cell level in the leaf mesophyll tissues undergoing senescence.
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