We are indebted to Clemire Cimper for her excellent technical support. Work in the laboratory of H.L. was funded by Institut Pasteur, Centre National pour la Recherche Scientific, and the Agence Nationale de la Recherche (Laboratoire d&#39;Excellence Revive, Investissement d&#39;Avenir; ANR-10-LABX- 73), the Agence Nationale de la Recherche (ANR-16-CE13-0017-01) and Fondation ARC (PJA 20161205028). C.C. and A.C. are funded by the Ph.D. and postdoctoral fellowships from the Revive Consortium.

Animals were handled as per European Community guidelines and the ethics committee of the Institut Pasteur (CETEA) approved protocols.
1. Preparations of the Stock Solutions
<ol>
	<li>Prepare the materials for muscle sample fixation.Dissolve 0.5 g of tragacanth gum with 20 mL water at RT to make the freezing-embedding medium for muscle fixation.</li>
	<li>Prepare the solutions for SA-&#946;-Gal staining.
	<ol>
		<li>Prepare the stock solutions of K3Fe(CN)6 (100 mM), K4Fe(CN)6 (100 mM), MgCl2 (1M) by dissolving the respective powders in distilled water.</li>
		<li>Prepare the stock solution of 0.2% C20H6Br4Na2O5 (Eosin) by diluting it in water.</li>
		<li>Prepare the stock solution of C14H15BrClNO6(X-gal) (50 mg/mL) by dissolving the X-gal powder in C3H7NO (dimethylformamide, DMF).</li>
		<li>Store K3Fe(CN)6 and K4Fe(CN)6 solutions at 4 &#176;C, and MgCl2 at RT. 
		NOTE: X-gal can be stored in aliquot at -20 &#176;C, up to 6 months. Eosin solution can be kept at RT and reused after filtering if necessary. K3Fe(CN)6 and K4Fe(CN)6 solutions are protectedfromthe light. X-gal solution is not stable in water and has to be protected from light.</li>
	</ol>
	</li>
	<li>Preparation of the solutions for Nanog staining: the permeabilization solution contains 0.1% Na3C6H5O7 (trisodium citrate), 0.1% C14H22O(C2H4O)n(n = 9-10) (Triton X-100) in distilled water, which should be stored at 4 &#176;C. Prepare the blocking solution containing 5% fetal bovine serum (FBS) in phosphate-buffered saline (PBS), which should be stored at RT.</li>
</ol>
2. SA-&#946;-Gal Staining on Frozen TA Muscle Section
<ol>
	<li>Prepare the fixation material for the TA muscle by placing a small amount of the tragacanth gum on a slice of cork.</li>
	<li>Injured mice of both sexes (2 month old, C57/B6) were injured 10 days before with cardiotoxin (CDX) as described previously13. Use non-injured (PBS injection) TA from the same mouse as the negative control. If in vivo reprogramming is desired, treat each mouse with Dox (1 mg/mL) in the drinking water on the same day (protected from the light), right after CTX injury. 
	NOTE: The Dox solution needs to be changed every 3 days for a total treatment duration of 7 days.
	<ol>
		<li>Isolate both TA muscles (injured and control) from mice as described previously (Figure 1A)13. To ensure the transverse sections, insert the distal tendon of the TA muscle into the tragacanth gum and leave roughly &#190; part of the muscle outside (Figure 1B) and freeze directly in liquid nitrogen cooled isopentane for &#60;1 min. 
		NOTE: Make sure the TA muscle is in a perpendicular position and in the center of the cork. Samples can be stored at -80&#176;C or directly cryosectioned in 10 &#181;m sections.</li>
	</ol>
	</li>
	<li>Process the TA muscles as described in the Figure 1B.
	<ol>
		<li>Distribute the 1st-10th sections in the correct order onto ten different slides at the upper left position of each slide. Place the 11th section adjacent to the 1st section on the first slide; the 12th section will follow the same order to be placed right besides the 2nd section on the second slide. Repeat this process till 10 sections/slide are obtained for ten slides (100 sections in total) to ensure minimum 1 mm distance between the first section and the last section.</li>
	</ol>
	</li>
	<li>Fix the sections for 4 min in PBS containing 1% paraformaldehyde and 0.2% glutaraldehyde. Wash with PBS, 2x&#160;10 min. Next, incubate the sections in PBS (pH = 5.5) for 30 min. Perform all the steps at RT. 
	NOTE: The fixation has to be mild to maintain enzymatic activity. Perform this step under the hood. The pH of the PBS is critical and the X-gal solution must be protected from light.</li>
	<li>Incubate sections in the X-gal solution containing: 4 mM K3Fe(CN)6, 4 mM K4Fe(CN)6, 2 mM MgCl2, and 400 &#181;g/mL X-Gal in PBS, pH = 5.5. Incubate in the dark at 37 &#176;C for a minimum of 24 h. Wash the slides with PBS, 3x 10 min, at RT. 
	NOTE: The incubation requires a minimum of 24 hours and can last for 48 hours to maximize the SA-&#946;-Gal signal. The solution needs to be changed after 24 hours of incubation. The slides should be protected from light. If only SA-&#946;-Gal staining is desired, continue with step 2.5. If co-staining with Nanog is desired, please skip forward to step 3.1.</li>
	<li>Fix slides in 1% paraformaldehyde in PBS for 30 min. Wash slides with PBS, 3x 10 min. Counterstain with 0.2% eosin. Immerse the slides in the eosin solution for 1 min and rinse them with distilled water briefly. Finally, mount the slides with aqueous non-fluorescing mounting medium (see Table of Materials). 
	NOTE: It is essential to perform the post-fixation. Perform this step under the hood. All steps are performed at RT.</li>
</ol>
3. Immunohistochemistry Using Anti-Nanog Antibody
<ol>
	<li>Fix the slides with PBS containing 4% paraformaldehyde for 10 min. Wash with PBS, 2x 10 min. Add 200 &#181;L of the permeabilization solution directly onto the slides and incubate for 5 min.
	<ol>
		<li>Wash slides with PBS, 2x 5 min and in the last wash, use 200 &#181;L PBS containing 0.25% BSA directly on the slides. Perform all these steps at RT. 
		Caution: Perform the fixation step under the hood.</li>
	</ol>
	</li>
	<li>Incubate slides with the primary anti-Nanog antibody (final concentration: 1.25 ug/mL) overnight at 4 &#176;C in PBS containing 5% FBS. Wash slides with PBS, 2x 10 min and in the last wash, use 200 &#181;L PBS containing 0.25% BSA for 5 min. Perform all the washing steps at RT. 
	NOTE: Incubate slides in a box with wet paper towel to prevent evaporation.</li>
	<li>Incubate slides with 100 &#181;L of rAb-HRP secondary antibody from a ready to use kit (see Table of Materials) for 45 min. Wash slides with PBS, 3x 5 min, to remove secondary antibody. Perform all the steps at RT with protection from light.</li>
	<li>Visualization
	<ol>
		<li>First, dilute the 3,3&#39;-diaminobenzidine (C12H14N4C12H18Cl4N4(4HCl), DAB) in the substrate buffer provided by the ready to use kit (20 &#181;L of DAB for 1 mL of substrate buffer solution). Add 100 &#181;L of DAB solution directly onto each slide and incubate for a maximum of 10 min at RT. 
		NOTE: The diluted DAB solution should be prepared freshly and can be stored for up to one week at 4 &#176;C. The incubation time can be adjusted to minimize the background signal but must be kept the same for all slides.</li>
		<li>Remove the DAB solution by rinsing with water. Counterstain the slides with fast red solution (ready to use, see the Table of Materials) for 20 min. Wash the slides with water again briefly.</li>
		<li>Dehydrate them with 95% ethanol for 5 min followed by 100% ethanol, 2x 5 min. Finally, mount the slides with quick-hardening mounting medium (see Table of Materials).</li>
		<li>Observe the slides under the microscope in bright field at 20X to avoid background. 
		NOTE: Fast red solution can be kept at RT and re-used after filtering.</li>
	</ol>
	</li>
</ol>
4. Analysis and Quantification
<ol>
	<li>SA-&#946;-Gal quantification
	<ol>
		<li>Scan the slides and choose the best sections on each slide based on the acquired images. Use at least 2 sections/TA for the quantification. Judge the section&#39;s quality based on tissue integrity, quality of the staining, and counterstaining. For the quantification, choose the two highest quality sections with the maximum possible distance in between samples, which allows better representation of SA-&#946;-Gal quantification throughout the whole TA muscle.</li>
		<li>Quantification of SA-&#946;-Gal positive cells (Figure 2).
		<ol>
			<li>Determine the rank of the pixel size by manually selecting the smallest and the largest positive SA-&#946;-Gal cells.
			<ol>
				<li>Open the digital image of the scanned slide using ImageJ software.</li>
				<li>In the interface, click &#39;Analyze&#39; &#62; &#39;Tools&#39; &#62; &#39;ROI Manager&#39; &#62; &#39;Analyze&#39; &#62; &#39;set measurement&#39; &#62; &#39;select Area&#39;. Use the selection tool and surround the smallest and biggest positive SA-&#946;-Gal cell and add it to the ROI manager by clicking on &#39;Add&#39;. Measure the sizes using the &#39;Measure&#39; button and save the values for later use.</li>
			</ol>
			</li>
			<li>Adjust the threshold parameter to ensure all the visible positive cells are selected.
			<ol>
				<li>Convert the image to gray scale by clicking &#39;Image&#39; &#62; &#39;type&#39; &#62; &#39;8-bit&#39; (Figure 2A). Next, go to &#39;Image&#39; &#62; &#39;Adjust&#39; &#62; &#39;Threshold&#39;, move the second cursor until all the positive SA-&#946;-Gal cells are covered in red (Figure 2B), then click &#39;Apply&#39;.</li>
				<li>Analyze particles by clicking &#39;Analyze&#39; &#62; &#39;Analyze Particles&#39; &#62; &#39;Size (pixel^2)&#39;, and apply the value defined in step 4.1.2.1, enter &#39;Circularity&#39;: &#39;0.00-1.00&#39;, click &#39;ok&#39; ; a summary of all the counted particles is shown in the ROI manager (Figure 2D).</li>
			</ol>
			</li>
			<li>Transfer all the selected particles to the original image. Adjust the selection manually to ensure accurate quantification. Add positive cells (green arrow, Figure 2E) and/or remove false positive cells (red arrow, Figure 2E). Finally, measure the area by outlining the section (Figure 2D) using the &#39;selection tool&#39;. Click &#39;ROI manager&#39; &#62; &#39;Add&#39; &#62; &#39;Measure&#39;.</li>
			<li>Importantly, normalize the number of positive cells by the area of the section.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Nanog quantification
	<ol>
		<li>Count the Nanog positive cells under the microscope in the bright field manually at 20X magnification. 
		NOTE: The fast red counterstaining allows good evaluation of the morphology of the TA muscle. However, the staining is very light and lacks a clear outline of the section. The scanner often fails to identify the boundary of the sections and cannot focus correctly. It is possible to use marker to circle the edges of the sections before scanning or use a more sensitive scanner to detect the section. For the current protocol, it is most efficient to count the Nanog positive cells manually.</li>
	</ol>
	</li>
</ol>

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Y., Krtolica, A., Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+senescence-associated+secretory+phenotype:+the+dark+side+of+tumor+suppression.">The senescence-associated secretory phenotype: the dark side of tumor suppression.</a> Annu Rev Pathol. 5, 99-118 (2010).</li><li>Yun, M. H., Davaapil, H., Brockes, J. 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(119), (2017).</li><li>Le Roux, I., Konge, J., Le Cam, L., Flamant, P., Tajbakhsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+is+required+to+prevent+p53-dependent+senescence+following+skeletal+muscle+injury.">Numb is required to prevent p53-dependent senescence following skeletal muscle injury.</a> Nat Commun. 6, 8528 (2015).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nat Protoc. 4 (12), 1798-1806 (2009).</li><li>Dimri, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proc Natl Acad Sci U S A. 92 (20), 9363-9367 (1995).</li><li>Lee, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+beta-galactosidase+is+lysosomal+beta-galactosidase.">Senescence-associated beta-galactosidase is lysosomal beta-galactosidase.</a> Aging Cell. 5 (2), 187-195 (2006).</li><li>Krishna, D. 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The authors declare that they have no competing financial interests.

Here, we present a method to detect both senescent and pluripotent stem cells in the skeletal muscle of reprogrammable mice. This method could be used to evaluate and quantify both senescence and induce cellular plasticity in vivo, and examine the role of senescence in tissue repair and regeneration.
In the current protocol, the senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is used to detect in vivo senescent cells in the skeletal muscle. This assay detects...

Cellular senescence is a form of stress response characterized by a stable cell-cycle arrest. In the last decade, research has firmly established that senescence is associated with various biological and pathological processes including embryonic development, fibrosis, and organism ageing1,2. Cellular senescence was first identified in human fibroblasts at the end of their replicative lifespan triggered by telomere shortening3. Besides replicative stress, there are many other stimuli that can induce senescence, including DNA damage, oxidative stress, oncogenic signals, and genomic/epigenomic alterations, any of which may eventually activate the p53/p21 and/or pRB pathways to establish and reinforce the permanent growth arrest1. One of the important characteristics of senescent cells is that they remain metabolically active and robustly express a senescence-associated secretory phenotype (SASP): secretion of many inflammatory cytokines, growth factors, and extracellular matrix factors4. SASP factors have been proposed to play an important role in mediating and amplifying the senescence effect, due to their potent effects on attracting immune cells and altering local and systemic tissue milieus1. Interestingly, senescence has been recently proposed to be important for tissue repair and regeneration5,6. In addition, data from several labs, including ours, has suggested that tissue damage-induced senescence might enhance cellular plasticity, via SASPs, to promote regeneration7-9. Therefore, all the emerging data highlight the importance of studying senescence in vivo.
In the post induced pluripotent stem cell&#160;(iPSC) era, cellular plasticity is the capacity of a cell to acquire a new identity and to adopt an alternative fate when exposed to different stimuli both in culture and in vivo10. It is known that full reprogramming can be achieved in vivo11,12, where the expression of the the cassette containing four Yamanaka factors: Oct4, Sox2, Klf4, and c-Myc (OSKM) can be induced in vivo to promote teratomas formation in multiple organs. Therefore, a reprogrammable mouse model (i4F) can be used as a powerful system to identify critical regulators and pathways that are important for cellular plasticity11.
A suitable and sensitive in vivo system is essential to understand how cellular senescence regulates cellular plasticity in the context of tissue regeneration. Here, we present a robust system and a detailed protocol to evaluate the link between senescence and cellular plasticity in the context of tissue regeneration. The combination of cardiotoxin (CTX) induced muscle damage in the Tibialis Anterior (TA) muscle group, a well-established system to study tissue regeneration, and the i4F mouse model, allows the detection of both cellular senescence and in vivo reprogramming during muscle regeneration.
To evaluate the link between cellular plasticity and senescence, i4F mice are injured with CTX to induce acute muscle damage and treated with doxycycline (0.2 mg/mL) over 7 days to induce in vivo reprogramming. While a CTX induced acute muscle damage and regeneration protocol has been recently published13, for ethical reasons, this procedure will be omitted in the current protocol. TA muscle samples will be collected at 10 days post injury13, when the peak of senescent cells have been previously observed14. Here, this detailed protocol describes all the steps required to evaluate the level of senescence (via SA-&#946;-Gal) and reprogramming (via IHC staining of Nanog).
Senescence-associated beta-galactosidase (SA-&#946;-Gal) assay is the most commonly used assay to detect senescent cells both in culture and in vivo15. Compared to other assays, the SA-&#946;-Gal assay allows the identification of the senescent cells in their native environment with intact tissue architecture, which is particularly important for in vivo study. Moreover, it is possible to couple the SA-&#946;-Gal assay with other markers using IHC. However, the SA-&#946;-Gal assay does require fresh or frozen samples, which remains a major limitation. When fresh or frozen tissues are routinely available, such as frozen TA muscle samples, SA-&#946;-Gal is obviously the most suitable assay to detect senescent cells. Nanog is the marker used to detect reprogramed cells for two reasons: 1) it is an essential marker for pluripotency; 2) more importantly, its expression is not driven by doxycycline (dox), therefore it detects induced pluripotency rather than the forced expression of the Yamanaka cassette.
It is important to note, the staining protocols presented in this study can be conducted separately to simplify the quantification procedure, but can also be done in a co-staining procedure to visualize both senescent and pluripotent stem cells on the same section.

<table>
	<tbody>
		<tr>
			<td>K3Fe(CN)6</td>
			<td>Sigma</td>
			<td>13746-66-2</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>K4Fe(CN)6</td>
			<td>Sigma</td>
			<td>14459-95-1</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>7786-30-3</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Sigma</td>
			<td>B4252</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D3447</td>
			<td>For inducing in vivo reprogramming</td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Lotaxan Valence, France</td>
			<td>L8102</td>
			<td>For muscle injury</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>111-30-8</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron microscopy science</td>
			<td>50-980-487</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>NaCitrate : Sodium Citrate monobasic bioxtra, anhydre</td>
			<td>Sigma</td>
			<td>18996-35-5</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma</td>
			<td>93443</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A3608</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Antibody anti- Nanog</td>
			<td>Cell signalling</td>
			<td>8822S</td>
			<td>Rabbit monoclonal antibody</td>
		</tr>
		<tr>
			<td>EnVision+ Kits (HRP. Rabbit. DAB+)</td>
			<td>Dako</td>
			<td>K4010</td>
			<td>For Nanog revelation</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Leica</td>
			<td>380159EOF</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Fast red</td>
			<td>Vector Laboratories</td>
			<td>H-3403</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Thermo Scientific Shandon Immu-Mount</td>
			<td>Fisher scientific</td>
			<td>9990402</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Quick-hardening mounting medium for microscopy : Eukitt&#174;</td>
			<td>Sigma</td>
			<td>25608-33-7</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Microscope for Nanog quantification</td>
		</tr>
		<tr>
			<td>Mouse: i4F-A</td>
			<td>Abad et al., 2013</td>
			<td>N/A</td>
			<td>Reprogrammable mouse model</td>
		</tr>
		<tr>
			<td>Skeletal muscle, Tibialis Anterior</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Zeiss</td>
			<td>Axio Scan Z1</td>
			<td>slides scanning</td>
		</tr>
	</tbody>
</table>

evaluation of injury-induced senescence and in vivo reprogramming in the skeletal muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Detecting muscle injury-induced cellular senescence
It has been recently demonstrated that muscle injury induces transient cellular senescence14. At 10 days post-injury (DPI), the majority of the damaged myofibers are undergoing regeneration with centrally located nuclei, a hallmark of regenerating myofibers, and the architecture of the muscle is re-established. The infiltrating inflammatory cells are dramat...

Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

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Research

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Developmental Biology

9.5K Views.  Institut Pasteur. Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo. 

Video: Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...