Terminal Transferase-Mediated dUTP Nick End-Labeling—TUNEL Assay: An In Situ Method to Detect DNA Fragmentation in Apoptotic Cells

Abstract

Source: Brannelly, L. A., et al. Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis. J. Vis. Exp. (2018).

In this video, the TUNEL assay is performed to detect the presence of apoptotic cells in a sample. The study involves using a special DNA polymerase - terminal deoxynucleotidyl transferase - that catalyzes the addition of labeled dUTPs to the 3' terminus of fragmented DNA, which can be observed and identified under a fluorescence microscope.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. TUNEL Assay

1. Euthanize animals that are showing clinical signs of chytridiomycosis and an equal number of Bd-negative control animals.
2. Dissect skin (dorsal, ventral, and thigh) samples from each animal. Fix the skin samples for 2 h in 4% v/v phosphate buffered formaldehyde. The short and consistent fixing time allows the tissues to be fully fixed, but also allows for effective and accurate immunohistochemistry staining. Then transfer to 80% ethanol until embedding f....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
POLARstar Omega	BMG Labtech		Luminescent plate reader
384 well flat clear bottom plate	Corning	3707
384 well low flange white flat bottom plate	Corning	3570
Formal-Fixx 10% Neutral Buffered Formalin	Fisher	6764254
ApopTag Red In Situ Apoptosis Detection Kit	Merck Millipore	S7165
PBS (Phosphate Buffered Saline),  pH 7.2 (1X)	Thermo/Life	20-012-043
Parafilm	Bemis	PM996
Ethanol, 200 Proof, Molecular Grade	Fisher	BP2818500
ZEISS Axio Scan florescent miscroscope	Carl Zeiss		Florescent microscope
Cell culture petri plates	Nunc	263991