This work was supported by NIH-NINDS grant RO1-NS065895 and NIH-NINDS grant 5-F31-NS076250-02. 
We thank JHU SOM Microscope Facility for the use of the cryostat.

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
<ol>
	<li>Sacrifice a neonatal mouse by CO2 inhalation.</li>
	<li>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 subsequently section on a cryostat.</li>
	<li>Place the hindlimb in 10&#160;ml of ice cold 4% paraformaldehyde in phosphate-buffered saline (PBS) in a 15&#160;ml tube.</li>
	<li>Immersion fix the hindlimb in paraformaldehyde for 4-24&#160;hr at 4&#160;&#176;C. Embryonic tissue requires only several hours of fixation.
	<ol>
		<li>Avoid fixation longer than 24&#160;hr, as this may reduce TUNEL assay efficiency.</li>
	</ol>
	</li>
	<li>Cryoprotect the hindlimb by immersing it in sucrose solution.
	<ol>
		<li>Change paraformaldehyde to 10% sucrose in PBS in the same 15&#160;ml tube, incubate overnight at 4&#160;&#176;C.</li>
		<li>Change to 30% sucrose in PBS, incubate overnight at 4&#160;&#176;C or until the hindlimb sinks to the bottom of the tube. Tissue may be left in 30% sucrose for a longer period, provided that the sucrose has been sterile-filtered.</li>
	</ol>
	</li>
</ol>
2. Tissue Embedding
<ol>
	<li>Fill a labeled embedding mold half-way with embedding medium.</li>
	<li>Place hindlimb on top of the embedding medium, with the cut side as close as possible to one wall of the mold. Mark the orientation of the hindlimb on the outside of the mold.
	<ol>
		<li>Embed several hindlimbs and cut in the same block. However, it is important to ensure that all hindlimbs in the block are oriented parallel to each other to get sections at matching levels along the axis of the limb.</li>
	</ol>
	</li>
	<li>Cover hindlimbs with embedding medium. If hindlimbs shift while pouring the medium, re-orient them with blunt-tipped forceps or a toothpick.</li>
	<li>Allow embedding medium to infiltrate the tissue at room temperature for at least 15&#160;min.</li>
	<li>Place molds directly on dry ice or in isopentane on dry ice to freeze embedding medium. Make sure to keep the molds level at all times and avoid shifting of the embedded tissue.</li>
	<li>Keep frozen molds at -80&#160;&#176;C until ready to cut, for up to several months.</li>
</ol>
3. Cryosectioning Embedded Limbs
<ol>
	<li>Set cryostat chamber temperature to -20&#160;&#176;C, object temperature to -18&#160;&#176;C; adjust object temperature down as necessary.</li>
	<li>Equilibrate the tissue block inside the cryostat chamber for at least 30&#160;min. Correct block temperature is crucial for section quality.</li>
	<li>Cut transverse sections at a thickness of 10&#160;&#956;m or less. Thicker sections will not be fully penetrated by TUNEL reagents. Use the anti-roll plate, blades, and blade angle (typically 5&#176;) recommended for the cryostat.</li>
	<li>Collect sections onto gelatin- or vectabond-coated glass slides at room temperature. Place immediately into a slide box on dry ice. Keep the slide box in a separate container with dry ice placed near the cryostat within arm&#8217;s reach.
	<ol>
		<li>Cut transverse serial sections through the long axis of the limb, skipping 100-200&#160;&#956;m. Collect at least 3 adjacent sections at each serial level.</li>
	</ol>
	</li>
</ol>
4. TUNEL Assay on Hindlimb Sections Using a Commercially Available Kit (all steps at room temperature unless noted)
<ol>
	<li>Thaw slides with sections to room temperature and dry overnight or over the weekend.</li>
	<li>Rehydrate sections by immersing slides in PBS for 2&#160;x&#160;10&#160;min in a Coplin jar. Dry with a lab wipe, removing as much liquid around the section as possible.</li>
	<li>Permeabilize sections
	<ol>
		<li>Use either a non-proteolytic, saponin-based commercial reagent or 0.5% Tween20 and 0.05% Triton X100 in PBS.</li>
		<li>Place the slide into a humidity chamber. A simple humidity chamber is an empty pipette tip box with a tight lid, with just enough water added to cover the bottom of the box.</li>
		<li>Pipette enough reagent onto the slide to cover the section (50&#160;&#956;l is sufficient for neonatal mouse limbs). Do not pipette directly onto the section, as repeated agitation may cause the section to lift off the slide.</li>
		<li>Using blunt-tipped forceps, place a small rectangle of parafilm on top of the section to spread out the drop of reagent and completely cover the section. If any bubbles form under the parafilm, gently lift it off, dry, and reapply.</li>
		<li>Incubate slides with permeabilization reagent for 1&#160;hr in a covered humidity chamber. If sections begin to lift off from the slide, permeabilization time may be reduced to 30&#160;min.</li>
		<li>Wash slides in PBS in a Coplin jar 2&#160;x&#160;5&#160;min. The parafilm rectangles should float to the top and can then be removed and discarded.</li>
		<li>Using a lab wipe, dry as much liquid around the sections as possible.</li>
	</ol>
	</li>
	<li>TdT-mediated DNA break labeling
	<ol>
		<li>Follow the kit instructions to make 1x TdT labeling buffer, pipette enough labeling buffer onto the slide to cover tissue section (50&#160;&#956;l is sufficient for neonatal mouse limbs). Incubate in the humidity chamber for 5&#160;min. Alternatively, immerse the slide in TdT labeling buffer in a coplin jar.</li>
		<li>Follow kit instructions to make TdT labeling mix. The kit includes cobalt, magnesium, and manganese cations; the manganese cation works well on a variety of tissues, including skeletal muscle, nervous system tissues, liver, skin, and bone.</li>
		<li>For a positive control, add DNase (labeled &#8220;nuclease&#8221; in the kit) to the TdT labeling mix. Alternatively, pre-treat a positive control section with DNase in nuclease buffer for 1&#160;hr at 37&#160;&#176;C, according to kit instructions. Keep DNase solution and DNase-treated sections away from other experimental sections to prevent cross-contamination.</li>
		<li>For a negative control, omit TdT enzyme from the labeling mix.</li>
		<li>Use a lab wipe to remove as much TdT labeling buffer from the slide as possible. Pipette TdT labeling mix onto the slide (50&#160;&#956;l per section), cover with a new rectangle of parafilm, and incubate in the humidity chamber at 37&#160;&#176;C for 1&#160;hr.</li>
		<li>Follow the kit instructions to make 1x stop buffer. Use a lab wipe to remove TdT labeling mix from the slide, and add 200-300&#160;&#956;l stop buffer to cover the tissue section, and incubate in the humidity chamber for 5&#160;min. Alternatively, immerse the slide in stop buffer in a Coplin jar.</li>
		<li>Wash the slide 2&#160;x&#160;2&#160;min in PBS. Use a lab wipe to remove as much PBS from the slide as possible.</li>
	</ol>
	</li>
	<li>Fluorescent labeling
	<ol>
		<li>Follow kit instructions to prepare fluorescein labeling solution to label dNTPs at DNA breaks. Pipette solution onto the slide (50&#160;&#956;l per section), cover with a new rectangle of parafilm, and incubate in the humidity chamber for 20&#160;min.</li>
		<li>Wash slide for 2&#160;min in PBS in a Coplin jar. Use a lab wipe to remove as much PBS as possible from the slide.</li>
		<li>Dilute Hoechst 33258 nuclear stain to 1&#160;&#956;g/ml in PBS and pipette onto the slide (100-200&#160;&#956;l is sufficient to cover section). Incubate in the humidity chamber for 5&#160;min.</li>
		<li>Wash 3x for 5&#160;min in PBS.</li>
		<li>Coverslip with antifade fluorescence mounting media and seal edges with nail polish.</li>
	</ol>
	</li>
</ol>
5. Digital Image Acquisition
<ol>
	<li>Acquire images using a conventional fluorescent microscope with DAPI, FITC, and Texas Red filters (or equivalent). Use a higher magnification objective (20X or 40X) to verify that the labeling was successful and nuclei (intact or fragmented) were labeled. A 10X objective is sufficient for quantitation of TUNEL-positive puncta.</li>
	<li>Collect images of the Hoechst staining, TUNEL staining, and autofluorescent signal as three separate false-color channels combined in one image. If the TUNEL fluorophore label is green, image autofluorescent signal in the red filter, and vise versa.
	<ol>
		<li>Keep all imaging settings equivalent between slides for subsequent thresholding and quantitation. For each filter, preset and record exposure times, gain, and binning (no binning is best); do not use automatic exposure or gain settings.</li>
		<li>Use trial-and-error to find exposure and gain settings that will best capture the range of intensities across all stained slides. Ensure that there are no saturated pixels in the acquired images, as this will bias quantitation.</li>
	</ol>
	</li>
	<li>Take images of the entire muscle group of interest. This may require multiple images to be &#8220;stitched&#8221; together.</li>
</ol>
6. Image Analysis
<ol>
	<li>Use commercially available image analysis programs to analyze digital images of TUNEL staining. Analysis using commercial software is shown here. Alternatively, the free software ImageJ from NIH can be used to analyze TUNEL staining.</li>
	<li>Stain a section adjacent to the TUNEL-stained section with hematoxylin and eosin (H&#38;E) and take a full-section digital image with a conventional brightfield microscope. Use this H&#38;E image in combination with an anatomy atlas5,18,19 to trace the anatomical structures to be quantified.</li>
	<li>With the TUNEL channel turned off, manually trace the outline of the area to be quantified, using Hoechst and autofluorescence channels for guidance. Convert the traced area into a mask (a set of selected pixel coordinates to be analyzed).</li>
	<li>Turn on the TUNEL channel. Using the intensity thresholding function in the image analysis program, set the lower threshold to exclude all autofluorescence signal.</li>
	<li>Apply the same threshold settings to all images in the study set. Convert thresholded selections into masks.</li>
	<li>For each image, combine the muscle area mask and the TUNEL mask. The new combination mask will represent TUNEL signal within the traced muscle area only.</li>
	<li>Perform mask quantitation on the combination mask to determine number of objects and object area of the TUNEL signal within the traced muscle area. Perform this using the Particle Analyzer function in ImageJ.</li>
</ol>

The authors have nothing to disclose.

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 should be aware of potential pitfalls. Tissue autofluorescence is a major concern in fluorescence imaging. In unperfused animals, remaining red blood cells will contribute fairly high autofluorescent signal. Endothelial cells may also be significantly autofluorescent. Therefore, it is important to confirm that autofluorescence is not mistaken for TUNEL-positive signal. This assessment is easily accomplished by taking an image in a channel with no fluorophore, to be used for comparison and possible subtraction from the TUNEL-positive channel. In this way, the fluorescence-based TUNEL method is superior to colorimetric brightfield microscopy methods that do not offer an internal control for background staining.
Tissue fixation will have significant effects on the success of TUNEL labeling. It is best to minimize fixation time in paraformaldehyde, not to exceed 24&#160;hr. Embryonic and neonatal tissue will require fixation times significantly shorter than 24&#160;hr.
When TUNEL labeling is successful, TUNEL-positive signal should be much higher than background autofluorescence, so that signal can be easily isolated by intensity-threshold gating using image analysis software. Antigen retrieval using the standard protocol of heating in citrate buffer27,35 may slightly improve low TUNEL signal if heating time is kept to a minimum (5&#160;min), but this pretreatment tends to reduce TUNEL signal if heating is extended for longer periods (e.g. 20&#160;min at 95&#160;&#176;C). Antigen retrieval will increase background autofluorescence and false positives, and thus may negate any gains in signal-to-noise ratio from increased TUNEL signal.
The TUNEL signal measurements must be normalized either to the total muscle area quantitated or to the number of nuclei per area quantitated. Because TUNEL measures apoptotic nuclei, the ideal normalization would be total nuclei. However, even in relatively thin (10&#160;&#956;m) muscle sections, the myofibers and nuclei are so numerous and packed so closely, that it is impossible to automatically count Hoechst-stained nuclei by thresholding and particle separation algorithms. Manual counting is also very labor-intensive and inaccurate. The feasible alternative is to normalize to total muscle area.
The TUNEL assay, used with appropriate tissue processing techniques and positive and negative controls, is a relatively fast, reproducible, quantitative method for detecting DNA damage and cell death in tissue. It can be used to confirm apoptosis as a pathological mechanism, to identify affected cell types, and to assess the efficacy of therapeutic treatments. This procedure will be of value to researchers in the fields of skeletal muscle disease and injury, including SMA, ALS, muscular dystrophy, toxin-induced myopathy, and exercise physiology.

Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) is the process of using the TdT enzyme to attach dUTP to 3&#8217; ends of double and single-stranded DNA breaks12,23. 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 preparations7,23,27,40. TUNEL has been used to study ischemia-induced cell death of neurons6,14,29 and cardiomyocytes43,44, excitotoxic neuronal cell death30,31, and as a biomarker in arthritis treatment39. It has also been used as a prognostic factor and tumor cell marker in various human cancers2,3,15,32,37,38,42.
Alternative methods exist for DNA damage and cell death detection, but they have technical challenges and caveats. Southern blotting may be used to quantify DNA damage in whole tissue lysates7,9-11, but this method does not provide cellular-level resolution and is difficult to quantify. The comet assay is an alternative cell-based method that requires extracting preserved nuclei from cells4,20,28,36. Although the comet assay works well on cultured isolated cells, it is much more difficult to prepare intact nuclei from skeletal muscle tissue8,21. As with Southern blot, the comet assay does not provide cell-type-specific information from a whole muscle tissue homogenate. Another alternative to the TUNEL method is immunohistochemistry using antibodies against single-stranded DNA25,33,41 or against proteins that participate in DNA damage response and cell death pathways (e.g. p53, H2AX, and caspases)13,17,22,40. Such antibody-based methods require thorough characterization of antibodies and excellent antibody specificity to yield a high signal-to-background ratio. Even when specific antibodies exist, they may require denaturation of the target protein through antigen retrieval procedures34,35. As we discuss here, antigen retrieval in muscle tissue results in unacceptably high autofluorescence. Unlike the alternative methods, TUNEL achieves DNA damage detection with a high signal and low background, excellent specificity that can be tested with simple positive and negative controls, good tissue penetration that does not require antigen retrieval, and cellular-level resolution. In addition, the TUNEL method takes about 4 hours to complete, whereas alternative methods typically require overnight incubations.
We study skeletal muscle cell death in a mouse model of spinal muscular atrophy (SMA)10 that was generated by Hsieh-Li and colleagues16. To quantify apoptotic cells in the muscle, we have developed a method of tissue preparation, staining, and quantitation that works robustly across different skeletal muscle groups at different developmental time points in mice. We use a commercially available TUNEL-labeling kit and commercially available image analysis software. We have also successfully used the TUNEL assay in combination with immunofluorescent staining in the spinal cord10.
The methods described here are useful for investigators who want to assess tissue pathology,&#160;mechanisms&#160;of disease, mechanisms&#160;of aging, and developmental (pre- and post-natal) cell death in skeletal muscle. The TUNEL technique is especially useful for studies of DNA damage and repair and cell death in model systems where only a subset of cells is affected and cellular level resolution is necessary.
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.

<table border="0" cellpadding="0" cellspacing="0" width="916">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:460px;">Comments/Description</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">4% Paraformaldehyde in phosphate buffered saline</td>
			<td style="width:121px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">19202</td>
			<td style="width:460px;">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 &#176;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.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td style="width:460px;">Used for cryoprotecting tissue before freezing. Sucrose from any supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O.C.T. compound&#160;</td>
			<td style="width:121px;">Tissue-Tek</td>
			<td style="width:119px;">4583</td>
			<td>Embedding medium for cryosectioning.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cryostat</td>
			<td style="width:121px;">Leica</td>
			<td style="width:119px;">CM 3050S</td>
			<td style="width:460px;">A Leica CM3050S cryostat was used for the preparations described here. Any cryostat capable of cutting 10 &#956;m sections may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass slides, 25 x 75 x 1 mm</td>
			<td style="width:121px;">Fisher</td>
			<td style="width:119px;">12-552-3</td>
			<td>Slides from any supplier may be used.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Gelatin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">G-9391</td>
			<td style="width:460px;">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 &#176;C, dip slides for several seconds, and let dry. Gelatin from any supplier may be used. Alternatively, gelatin-precoated slides may be purchased.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Chromium(III) potassium sulfate dodecahydrate (chrome alum)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">243361</td>
			<td style="width:460px;">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.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectabond tissue adhesion reagent</td>
			<td style="width:121px;">Vector Labs</td>
			<td style="width:119px;">SP-1800</td>
			<td style="width:460px;">Optional substrate for better tissue adhesion to glass slides; gellatin-coated slides may be used instead.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween20</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P9416</td>
			<td style="width:460px;">A detergent used to permeabilize tissue. Tween20 from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X100</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td style="width:460px;">A detergent used to permeabilize tissue. Triton X100 from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TACS 2 TdT fluorescein in situ apoptosis detection kit</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>Commercial kit for fluorescence-based TUNEL labeling.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase/nuclease</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>(included with kit)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase/nuclease buffer</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>(included with kit)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x phosphate buffered saline (PBS), pH 7.4</td>
			<td style="width:121px;">Amresco</td>
			<td style="width:119px;">780</td>
			<td style="width:460px;">Make 1x PBS for washes and dilutions. PBS from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DNase-free water</td>
			<td style="width:121px;">Quality Biologicals</td>
			<td style="width:119px;">351-029-131</td>
			<td>Water from any supplier may be used.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hoechst 33258</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">94403</td>
			<td style="width:460px;">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.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Parafilm M</td>
			<td style="width:121px;">multiple</td>
			<td style="width:119px;">807</td>
			<td style="width:460px;">Any other hydrophobic film or cover slip may be used. Available from multiple suppliers.&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent microscope with digital camera</td>
			<td style="width:121px;">&#160;--</td>
			<td style="width:119px;">&#160;--</td>
			<td style="width:460px;">Any fluorescent microscope capable of digitally capturing red, green, and blue fluorescence in separate channels may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vectashield antifade media</td>
			<td style="width:121px;">Vector Labs</td>
			<td style="width:119px;">H-1000</td>
			<td>Antifade media from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass coverslips, No.1 thickness</td>
			<td style="width:121px;">Brain Research Labs</td>
			<td style="width:119px;">2222-1</td>
			<td style="width:460px;">Cover slips from any supplier may be used. The smallest size of 22x22 mm is sufficient for neonatal mouse leg sections.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nail polish</td>
			<td style="width:121px;">Ted Pella</td>
			<td style="width:119px;">114-8</td>
			<td style="width:460px;">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.&#160;</td>
		</tr>
	</tbody>
</table>

detection and analysis of dna damage in mouse skeletal muscle in situ using the tunel method

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&#8217; 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.

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 TUNEL-positive signal, distributed uniformly across all tissues in the section (Figure 1C). The negative control (TdT enzyme omitted from reaction) should yield low-intensity background and autofluorescent signal only (Figure 1D). Skin provides an internal positive control in whole leg sections, as this tissue has a high rate of normal apoptosis (Figure 1A).
Red blood cells, bone, and endothelial cells may contribute significant autofluorescent signal (Figure 2). It is advisable to image each section in a separate channel with no fluorescent labeling to ensure that autofluorescence is not mistaken for positive TUNEL signaling. True TUNEL-positive signal should have much higher intensity than autofluorescence, so that it can be isolated by setting image intensity thresholds (Figure 2A, middle row). Alternatively, the autofluorescent channel may be digitally subtracted from the TUNEL channel to yield TUNEL-positive signal (Figure 2A, bottom row).
The TUNEL labeling method described here has been used to quantify skeletal muscle cell death in a mouse model of SMA10. TUNEL labeling shows an increase in the number of apoptotic profiles in leg muscles from 5 day old SMA mice, compared with littermate controls (Figure 3A). The increase in apoptosis is quantifiable by the method described above, indicating statistically significant differences in multiple muscle groups (Figure 3B).
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/52211/52211fig1highres.jpg" width="600" /> 
Figure 1: Representative TUNEL labeling of mouse hindlimb muscles. (A) TUNEL-labeled transverse section of mouse hindlimb showing tibialis anterior muscle, tibia, and skin. TA &#8211; tibialis anterior, Fdl &#8211; flexor digitorum longus, Edl &#8211; extensor digitorum longus. Green &#8211; TUNEL, blue &#8211; nuclei, red &#8211; autofluorescent signal. Dotted lines delineate muscle areas for quantitation. Skin adjoining hindlimb muscles provides an internal positive control for TUNEL labeling. Scale bar = 200&#160;&#956;m. (B) Representative high-magnification images identifying TUNEL-positive structures (green) in embryonic day 13 mouse skeletal muscle as apoptotic nuclei. Scale bar = 10&#160;&#956;m. A and B modified from Fayzullina and Martin 201410. (C) Transverse section of mouse hindlimb TUNEL-labeled after DNase treatment to serve as a positive control. Most nuclei have bright TUNEL labeling. Scale bar = 50&#160;&#956;m. (D) Transverse section of the same mouse hindlimb as shown in C (semi-adjacent to section in C) TUNEL-labeled with TdT enzyme omitted to serve as a negative control. There is no bright TUNEL-positive signal; the only green signal is autofluorescence as detected by colocalization with signal in the red channel. Scale bar = 50&#160;&#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52211/52211fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/52211/52211fig2highres.jpg" width="600" /> 
Figure 2: Tissue autofluorescence must be considered and excluded from quantitation when analyzing TUNEL labeling. (A) Mouse hindlimb transverse section with TUNEL labeling and autofluorescence: original image (top row), same image with adjusted thresholds (middle row), and same image with autofluorescence (red) channel subtracted (bottom row). Thresholding eliminates most red blood cell and endothelial cell autofluorescence but not the staining artifact (arrowhead, middle row). Channel subtraction also eliminates the staining artifact (arrowhead, bottom row). Green - TUNEL, red &#8211; autofluorescence, blue - nuclei. Scale bar = 100&#160;&#956;m. Gray rectangle delineates area magnified in B. (B) Higher magnification image of red autofluorescence panel in A. Red blood cells and some endothelial cells (arrows), and a tissue staining artifact (arrowhead) autofluoresce in both red and green fluorescence filters. Scale bar = 50&#160;&#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52211/52211fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/52211/52211fig3highres.jpg" width="600" /> 
Figure 3: TUNEL labeling shows cell death in the skeletal muscle of neonatal SMA mice. Lower hindlimb muscles from SMA mice at postnatal day 5 were labeled by the TUNEL method. (A) TUNEL labeling shows multiple apoptotic profiles in multiple muscle groups in SMA mouse muscle (right) compared with littermate control (left). Green &#8211; TUNEL, blue &#8211; Hoechst (nuclei), red &#8211; autofluorescence. Dotted lines delineate the muscle areas quantified. TA &#8211; tibialis anterior, Fdl &#8211; flexor digitorum longus, Edl &#8211; extensor digitorum longus. Scale bars = 200&#160;&#956;m. (B) TUNEL labeling was quantified as total pixel area of TUNEL signal normalized to total pixel area of muscle. Means &#177; standard errors are shown, n = 4 - 5 mice. Statistical significance between SMA and control for each muscle group (one-tailed t-test): * p &#60;0.05, ** p &#60;0.01, *** p &#60;0.001. This figure has been modified from Fayzullina and Martin 201410. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52211/52211fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method at JoVE.com

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

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.

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

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

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Nanyang Technological University

Research

JoVE Journal

Biology

16.2K Views.  Johns Hopkins School of Medicine. 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. 

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

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

Summary

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Chapters in this video

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Purification of Progenitors from Skeletal Muscle

Procedures for Rat in situ Skeletal Muscle Contractile Properties

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method

Electroporation of Plasmid DNA into Mouse Skeletal Muscle