Detection and analysis of DNA damage in mouse skeletal muscle in situ using the TUNEL method

Saniya Fayzullina, Lee J Martin

Research output: Contribution to journalArticle

Abstract

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3' ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Original languageEnglish (US)
JournalJournal of Visualized Experiments
Issue number94
DOIs
StatePublished - 2014

Fingerprint

Labeling
DNA Damage
Muscle
Skeletal Muscle
DNA
Tissue
DNA Nucleotidylexotransferase
Cell death
Processing
Fluorescence
Assays
Apoptosis
Enzymes
Single-Stranded DNA Breaks
Double-Stranded DNA Breaks
Dissection
Signal-To-Noise Ratio
deoxyuridine triphosphate
Single-Stranded DNA
Signal detection

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Detection and analysis of DNA damage in mouse skeletal muscle in situ using the TUNEL method. / Fayzullina, Saniya; Martin, Lee J.

In: Journal of Visualized Experiments, No. 94, 2014.

Research output: Contribution to journalArticle

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