Name: in situ immunofluorescent staining of autophagy in muscle stem cells
Uploaded: 2017-06-12T15:00:00
Description: Watch this Scientific Journal Video about In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells at JoVE.com

This work was supported by NIAMS AR064873, Epigen Project PB. P01.001.019/Progetto Bandiera Epigenomica IFT to L.L.

Mice were bred and maintained according to the standard animal facility procedures, and all experimental protocols were approved by the Animal Welfare Assurance and the internal Animal Research Ethical Committee according to the Italian Ministry of Health and complied with the NIH Guide for the Care and Use of Laboratory Animals.
1. Muscle Injury and the In Vivo Block of Autophagic Flux
<ol>
	<li>Muscle injury.
	<ol>
		<li>To induce acute skeletal muscle injury, inj...

<ol>
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C., 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=Skeletal+muscle+stem+cells:+effects+of+aging+and+metabolism+on+muscle+regenerative+function.">Skeletal muscle stem cells: effects of aging and metabolism on muscle regenerative function.</a> Cold Spring Harb Symp Quant Biol. 76, 101-111 (2011).</li><li>Cheung, T. H., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+stem+cell+quiescence.">Molecular regulation of stem cell quiescence.</a> Nat Rev Mol Cell Biol. 14 (6), 329-340 (2013).</li><li>Collins, C. A., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+the+adult+skeletal+muscle+satellite+cell.">Self-renewal of the adult skeletal muscle satellite cell.</a> Cell Cycle. 4 (10), 1338-1341 (2005).</li><li>Bentzinger, C. F., 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=Cellular+dynamics+in+the+muscle+satellite+cell+niche.">Cellular dynamics in the muscle satellite cell niche.</a> EMBO Rep. 14 (12), 1062-1072 (2013).</li><li>Bernet, J. D., 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=p38+MAPK+signaling+underlies+a+cell-autonomous+loss+of+stem+cell+self-renewal+in+skeletal+muscle+of+aged+mice.">p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice.</a> Nat Med. 20 (3), 265-271 (2014).</li><li>Cosgrove, B. D., 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=Rejuvenation+of+the+muscle+stem+cell+population+restores+strength+to+injured+aged+muscles.">Rejuvenation of the muscle stem cell population restores strength to injured aged muscles.</a> Nat Med. 20 (3), 255-264 (2014).</li><li>Sousa-Victor, 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=Geriatric+muscle+stem+cells+switch+reversible+quiescence+into+senescence.">Geriatric muscle stem cells switch reversible quiescence into senescence.</a> Nature. 506 (7488), 316-321 (2014).</li><li>Madaro, L., Latella, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forever+young:+rejuvenating+muscle+satellite+cells.">Forever young: rejuvenating muscle satellite cells.</a> Front Aging Neurosci. 7, 37 (2015).</li><li>Price, F. 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This protocol describes how to monitor autophagy in skeletal muscle stem cells during compensatory muscle regeneration. Several antibodies for the co-staining of LC3 and MyoD were tried, and the ones that work in mouse tissue sections and create successful results are listed here (see Materials Table). The permeabilization with methanol (see step 3.2.2) is highly recommended for successful staining.
The limitation of this protocol is linked to the intrinsic variability of the ...

Skeletal muscle regeneration is the result of the interaction between adult stem cells (Muscle Satellite Cells, MuSCs) and other cell types that are involved in the regenerative process. Muscle homeostasis and functionality are maintained by the combined signals arising from the muscle niche and systemic cues1,2. Throughout the lifetime, changes in the MuSC functionality, the muscle niche, and the systemic cues have been reported, leading to the decline of functional capacities in the elderly3. MuSCs are set in a niche beneath the basal lamina and, upon muscle injury, are activated to repair damaged muscles4,5. In order to ensure a productive regenerative response, it is crucial that MuSCs coordinate different processes necessary for the exit from quiescence, the self-renewal, and the proliferative expansion stage followed by the myogenic differentiation6. In the elderly and in muscular chronic diseases, all these functions are compromised, leading to altered muscle functionality2,3,6,7,8,9,10,11,12,13.
Macroautophagy (referred hereafter as autophagy) is emerging as a crucial biological process essential to preserve tissue homeostasis14. The autophagic process encloses trafficking mechanisms, where portions of cytoplasm, organelles, and proteins are engulfed into vesicles that eventually are degraded via the lysosome pathway, promoting the removal of toxic molecules and the recycling of macromolecules. This provides energy-rich compounds to support cell and tissue adaptation under stress or other adverse conditions15,16. Together with its cell-survival activity, autophagy can also work as a cell-death inducer, depending upon cell tissue context (e.g., normal versus cancer tissue) and the type of stress stimulus17,18.
Recent evidence indicates that autophagy is required to maintain muscle mass and myofiber integrity19,20 and has been reported to be impaired in different muscle dystrophies21,22,23, including Duchenne Muscular Dystrophy (DMD)24,25,26,27,28,29,30.Likewise, a progressive reduction of the autophagic process has been observed in the elderly31,32,33,34,35, after a loss of muscle mass (referred as sarcopenia)32,33,34,35,36,37, and in myofiber survival38.
A close relationship between autophagy and the regenerative potential of skeletal muscles was anticipated by a study from Wagers&#39;s laboratory, which showed that a calorie restriction enhances MuSC availability and activity39. This notion was further supported by the recent observation that the Foxo3-Notch axis activates the autophagic process during self-renewal40 and the MuSC transition from the quiescent to the proliferating state41. These data agree with the progressive reduction of basal autophagy from young to old and geriatric MuSCs, in association with the numerical and functional decline of MuSCs during ageing42.
In a recent paper, we demonstrated a close relationship between autophagy and the compensatory muscle regeneration that distinguishes the early stages of DMD progression. Accordingly, we observed a reduced autophagic flux at later stages of disease progression, when muscle regeneration is compromised and fibrotic tissue deposition occurs. Intriguingly, we showed that, in regenerating conditions, autophagy is activated in MuSCs and that modulating the autophagic process impacts MuSC activation and functionality30.
Altogether, these data highlight the urgency to explore the autophagic process in MuSCs during muscle regeneration in normal and pathological conditions and throughout the life span. Here, we provide a protocol to monitor the autophagic process in MuSCs in muscle regenerative conditions by performing in situ immunostaining for microtubule-associated protein 1A/1B-light chain 3 (LC3), a marker of autophagy43, and MyoD, a marker of myogenic lineage, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC, which plays a key role in orchestrating muscle regeneration.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:332px;">C57BL/6J</td>
			<td style="width:251px;">The Jackson Laboratory</td>
			<td style="width:128px;">000664</td>
			<td style="width:196px;">WT mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cardiotoxin 1</td>
			<td>Latoxan</td>
			<td>L8102</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Millex-VV</td>
			<td>Merck Millipore</td>
			<td>SLVV033RS</td>
			<td style="width:196px;">Syringe Filter Unit, 0.1 &#181;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Chloroquine diphosphate salt</td>
			<td>Sigma-Aldrich</td>
			<td>C6628</td>
			<td style="width:196px;">Caution: 
			Harmful if swallowed</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">BD Micro-Fine + 0,5 mL</td>
			<td>BD</td>
			<td>324825</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue-Tek O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue-Tek Cryomold Intermediate</td>
			<td>Sakura Finetek</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2-Methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>277258</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hematoxylin Solution, Harris Modified</td>
			<td>Sigma-Aldrich</td>
			<td>HHS32</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Eosin Y solution, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr height="122">
			<td height="122" style="height:122px;">o-Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>X1040</td>
			<td style="width:196px;">Caution: 
			Flammable liquid and vapour; May be fatal if swallowed and enters airways; Harmful in contact with skin; May cause respiratory irritation; Causes serious eye irritation</td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td style="width:196px;">Caution: 
			Flammable solid; Harmful if swallowed; Causes skin irritation; May cause an allergic skin reaction; Causes serious eye damage; May cause respiratory irritation; Suspected of causing cancer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DPBS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Eukitt - Quick-hardening mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-007-003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LC3B Antibody</td>
			<td>Cell signaling Technology</td>
			<td>2775</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:332px;">Monoclonal mouse anti-MyoD 
			(concentrated) clone 5.8A</td>
			<td>DAKO - Agilent Pathology Solutions</td>
			<td>M3512</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Laminin-2 (&#945;-2-chain) monoclonal antibody</td>
			<td>Enzo Life Sciences</td>
			<td>4H8-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A11008</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor 594 Goat Anti-Mouse IgG (H+L)</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A11005</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">Alexa Fluor Goat Anti-Rat IgM Antibody</td>
			<td style="width:251px;">Life technologies</td>
			<td style="width:128px;">A21248</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:332px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ immunofluorescent staining of autophagy in muscle stem cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

This protocol describes an efficient in situ method to detect autophagy in MuSCs during muscle regeneration.
CTX In Vivo Treatments:
Use CTX to induce muscle damage in TA muscles and use unperturbed muscles as controls. Since autophagy is highly dynamic, block the autophagic flux by performing IP injections of CLQ (Figure 1)...

Watch this Scientific Journal Video about In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells at JoVE.com

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Research

JoVE Journal

Biology

Procedures for Rat in situ Skeletal Muscle Contractile Properties

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 ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...