TUNEL Assay for Muscle Cell Apoptosis

In situ detection of cells with DNA strand breaks was performed on paraformaldehyde-fixed, paraffin-embedded muscle sections by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique, using an ApopTag-peroxidase kit (Chemicon International, Inc., San Francisco, CA) [37 (link),43 (link)]. Counting of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (DNT-DUT) nick end labeling-positive nuclei was carried out using an American Optical Microscope with a 40X objective and a pair of 10X eyepieces. A square grid fitted within one eyepiece provided a reference of 62,500 μm². The rate of muscle cell apoptosis was expressed as the percentage of the terminal (DNT-DUT) nick end labeling-positive apoptotic nuclei per total nuclei (apoptotic plus nonapoptotic) present within the reference area.

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Tsao J., Kovanecz I., Awadalla N., Gelfand R., Sinha-Hikim I., White R, & Gonzalez-Cadavid N. (2016). Muscle Derived Stem Cells Stimulate Muscle Myofiber Repair and Counteract Fat Infiltration in a Diabetic Mouse Model of Critical Limb Ischemia. Journal of stem cell research & therapy, 6(12), 370.

Publication 2016

ApopTag-peroxidase kit

Apoptotic Cells Deoxynucleotidyltransferase Deoxyuridine triphosphate Dna breaks Embedded paraffin Muscle Muscle cell Nuclei Optical microscope Paraformaldehyde Peroxidase

Corresponding Organization :

Other organizations : Charles R. Drew University of Medicine and Science, Harbor–UCLA Medical Center, The Lundquist Institute

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Percentage of terminal (DNT-DUT) nick end labeling-positive apoptotic nuclei per total nuclei (apoptotic plus nonapoptotic) present within the reference area

control variables

Paraformaldehyde-fixed, paraffin-embedded muscle sections
TUNEL technique using an ApopTag-peroxidase kit
Counting of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (DNT-DUT) nick end labeling-positive nuclei using an American Optical Microscope with a 40X objective and a pair of 10X eyepieces
A square grid fitted within one eyepiece providing a reference of 62,500 μm^2

positive controls

None mentioned

negative controls

None mentioned

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