Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Summary

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

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.

Introduction

Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) is the process of using the TdT enzyme to attach dUTP to 3’ ends of double and single-stranded DNA breaks^12,23. The TUNEL method for detection of apoptosis and DNA damage was first reported over 20 years ago by Gavrieli et al.^1,12,24. It has since been evaluated and optimized in different tissue preparations^7,23,27,40. TUNEL has been used to study ischemia-induced cell death of neurons^6,14,29 and cardiomyocytes^43,44, excitotoxic neuronal cell death^30,31, and as a biomarker in arthritis treatment³⁹. It ha....

Protocol

NOTE: All animal procedures described in this protocol were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health²⁶. The protocol (MO13M391) was approved by the Johns Hopkins University Animal Care and Use Committee.

1. Neonatal Mouse Sacrifice, Dissection, and Fixation

1. Sacrifice a neonatal mouse by CO₂ inhalation.
2. Immediately cut off the hindlimb above the knee. Until approximately postnatal day 7, the leg bones are soft enough to cut through with large laboratory scissors or sharp blade and to subs....

Results

With successful staining, TUNEL-positive signal should be bright enough to isolate from autofluorescence by setting intensity thresholds. TUNEL-positive objects at low magnification may appear as bright irregular fragments in skeletal muscle (Figure 1A). However, at higher magnification, some TUNEL-positive objects with the classic apoptotic morphology should be observed, if the cell death type involved is apoptosis (Figure 1B). The positive control (DNase added) should exhibit abundant .......

Discussion

A method to detect and quantitatively analyze DNA damage-associated apoptosis in mouse skeletal muscle is described. The procedure includes tissue harvesting, TUNEL staining, digital image acquisition, and image analysis. Common histological supplies and tools are needed, and a special commercial TUNEL kit is necessary. The essential big equipment items needed are a cryostat, epifluorescent microscope with digital image capability, and a computer system for image analysis.

The experimenter sho.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde in phosphate buffered saline	Electron Microscopy Sciences	19202	For procedures described here, 4% solution was prepared fresh from powder. Paraformaldehyde from any supplier may be used. Prepared formaldehyde solution should be stored at 4 °C and should not be used after its expiration date (up to several months). Paraformaldehyde is a carcinogen and a toxin by inhalation and skin contact. Please follow precautions specified in the MSDS when handling paraformaldehyde.
Sucrose	Sigma	S0389	Used for cryoprotecting tissue before freezing. Sucrose from any supplier may be used.
O.C.T. compound	Tissue-Tek	4583	Embedding medium for cryosectioning.
Cryostat	Leica	CM 3050S	A Leica CM3050S cryostat was used for the preparations described here. Any cryostat capable of cutting 10 μm sections may be used.
Glass slides, 25 x 75 x 1 mm	Fisher	12-552-3	Slides from any supplier may be used.
Gelatin	Sigma	G-9391	Gelatin is used to promote tissue section adhesion to glass slides. To coat glass slides with gelatin, dissolve 2.75 g gelatin and 0.275 g chrome alum in 500 ml distilled water, warm to 60 °C, dip slides for several seconds, and let dry. Gelatin from any supplier may be used. Alternatively, gelatin-precoated slides may be purchased.
Chromium(III) potassium sulfate dodecahydrate (chrome alum)	Sigma	243361	Chrome alum is added to gelatin solution to promote tissue adhesion on glass slides. It is a possible carcinogen and a toxin by inhalation and skin contact. Please follow precautions specified in the MSDS when handling chrome alum.
Vectabond tissue adhesion reagent	Vector Labs	SP-1800	Optional substrate for better tissue adhesion to glass slides; gelatin-coated slides may be used instead.
Tween20	Sigma	P9416	A detergent used to permeabilize tissue. Tween20 from any supplier may be used.
Triton X100	Sigma	T8787	A detergent used to permeabilize tissue. Triton X100 from any supplier may be used.
TACS 2 TdT fluorescein in situ apoptosis detection kit	Trevigen	4812-30-K	Commercial kit for fluorescence-based TUNEL labeling.
DNase/nuclease	Trevigen	4812-30-K	(included with kit)
DNase/nuclease buffer	Trevigen	4812-30-K	(included with kit)
10x phosphate buffered saline (PBS), pH 7.4	Amresco	780	Make 1x PBS for washes and dilutions. PBS from any supplier may be used.
DNase-free water	Quality Biologicals	351-029-131	Water from any supplier may be used.
Hoechst 33258	Sigma	94403	Nuclear dye. Any blue fluorescent nuclear dye may be used. As a DNA-binding dye, Hoechst is a suspected carcinogen and should be handled with protective equipment to minimize skin contact.
Parafilm M	multiple	807	Any other hydrophobic film or cover slip may be used. Available from multiple suppliers.
Fluorescent microscope with digital camera	--	--	Any fluorescent microscope capable of digitally capturing red, green, and blue fluorescence in separate channels may be used.
Vectashield antifade media	Vector Labs	H-1000	Antifade media from any supplier may be used.
glass coverslips, No.1 thickness	Brain Research Labs	2222-1	Cover slips from any supplier may be used. The smallest size of 22 x 22 mm is sufficient for neonatal mouse leg sections.
Nail polish	Ted Pella	114-8	Used to seal coverslips. Nail polish from any supplier (including regular retailers) may be used. Avoid using nail polish with color or additives that may reflect light during fluorescent imaging.