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>
	<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=Differential+response+of+skeletal+muscles+to+mTORC1+signaling+during+atrophy+and+hypertrophy.">Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy.</a> Skelet Muscle. 3 (1), (2013).</li><li>Chakkalakal, J. V., 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=The+aged+niche+disrupts+muscle+stem+cell+quiescence.">The aged niche disrupts muscle stem cell quiescence.</a> Nature. 490 (7420), 355-360 (2012).</li><li>Jang, Y. 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. 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=Inhibition+of+JAK-STAT+signaling+stimulates+adult+satellite+cell+function.">Inhibition of JAK-STAT signaling stimulates adult satellite cell function.</a> Nat Med. 20 (10), 1174-1181 (2014).</li><li>Tierney, M. T., 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=STAT3+signaling+controls+satellite+cell+expansion+and+skeletal+muscle+repair.">STAT3 signaling controls satellite cell expansion and skeletal muscle repair.</a> Nat Med. 20 (10), 1182-1186 (2014).</li><li>Judson, R. N., Zhang, R. H., Rossi, 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 ...

The overall goal of this immunofluorescent staining protocol is to monitor the autophagic process in muscle satellite cells during skeletal muscle regeneration. This method can help answer key questions in the tissue regeneration field about understanding the contribution of autophagy in deriving muscle regeneration and monitoring autophagy in satellite cells. The main advantage of this technique is that the autophagic process can be directly detected in satellite cells in tissue section from unperturbed and damaged muscles.Demonstrating this procedure will be Francesco Castagnetti, a great student from my laboratory. To induce acute skeletal muscle injury, load an insulin syringe, equipped with a 30 gauge needle, with 20 microliters of sterile filtered 10 micromolar cardiotoxin stock. Inject the cardiotoxin directly into the left tibialis anterior muscle of an equal number of male and female two month old, approximately 20 gram, C57 black six mice.To assess the autophagic flux, starting at 24 hours post injury, administer 50 milligrams per kilogram chloroquine every 24 hours for four days by intraperitoneal injection. Chloroquine treatment is critical as it leads to LC3 accumulation and detectability under active autophagic flux conditions. Five days after the injury, wet the animals with 70%ethanol.And use scissors to make a three millimeter perpendicular incision in the dorsal skin of the mouse at the hip level. Pull the skin towards the tail to expose the underlying muscles. And identify the tibialis anterior muscle.Use forceps to grasp the tibialis anterior muscle by the tendon. And carefully pull the muscle up toward the knee. Then pull the two distal tendons in opposite directions to separate the tibialis anterior muscle from the extensor digitorum longus muscle and cut the proximal tendon.Use the forceps to remove the thin fascia covering the muscle, taking care not to damage the tissue. And use a paper towel to remove any excess moisture from the sample. Place the excised muscle into the center of a tissue mold, containing optimal cutting temperature compound.And use pre-chilled forceps to carefully lower the mold into liquid nitrogen chilled isopentane for 20 to 30 seconds. Then store the frozen sample at negative 80 degrees celsius. After at least one night at negative 80 degrees celsius, set the cryostat between negative 15 to negative 23 degrees celsius and use a drop of optimal cutting temperature compound to attach the muscle sample to the cryostat chuck with the bulk of the tissue oriented at the top of the chuck.Then obtain seven to eight micrometer thick sections, collecting three to four tissue slices per slide. After all of the section have been acquired, air dry the tissue samples for 10 to 15 minutes, followed by hematoxylin and eosin staining. Check the quality of the sections under an optical microscope at a 10X magnification.And fix the samples with 4%paraformaldehyde in an incubation chamber. After 10 minutes, remove the fixative with four five to seven minute washes in PBS. Then use a hydrophobic pen to draw a barrier around the tissue sections.And cover the section with 200 microliters of negative 20 degree celsius methanol for a five minute incubation at negative 20 degrees celsius. Methanol permeabilization is critical as it facilitates a good retention of the LC3 staining. Next, wash the section four times in PBS as just demonstrated.And block the tissue samples with an unconjugated affinity purified anti-mouse IGG FAB fragment for at least 60 minutes at room temperature. At the end of the incubation, incubate the specimens in 100 microliters of primary antibody mix at four degrees celsius overnight. The next morning, wash the slides with 1%bovine serum albumin in PBS four times for 10 minutes per wash.Then incubate the samples with the appropriate secondary antibody cocktail for 45 minutes at room temperature protected from light. At the end of the secondary antibody incubation, wash the slides four times in PBS for five to seven minutes per wash. Followed by labeling with the second primary antibody cocktail for one to two hours at room temperature protected from light.Wash the sections in 1%BSA and PBS four times. Followed by incubation in the appropriate second antibody for 45 minutes at room temperature protected from light. Then wash the samples in PBS and label the tissues with 200 microliters of DAPI per slide for five minutes at room temperature protected from light.After one last set of PBS washes, remove the excess PBS from the slides and add 10 microliters of glycerol in PBS onto the center of each slide. Cover each slide with a coverslip, taking care to avoid bubbles. Select the 63X objective on a four laser confocal microscope integrated with an image capture system and analytical software.Center the fields of active regeneration on the first slide to allow manual focus of the regenerative areas. Then locate the myoD positive muscle satellite cells positioned within the laminin labeled myofibers. And obtain images within at least seven fields from at least three mice per experimental group.H and E staining reveals that while unperturbed muscles displayed generally invariable myofiber sizes, injured muscles typically featured disrupted myofibers that differ in size and are abundant in inflammatory infiltrate. Further, the central nucleation of the myofibers, that is absent in unperturbed muscles, is a sign of new fiber formation in mice undergoing active regeneration. MyoD positive muscle satellite cells express LC3 and laminin staining allows the proper localization of muscle cells in the niche beneath the basal lamina.While uninjured mice do not demonstrate any muscle satellite cell activation as evidenced by their lack of myoD positive cells and LC3 signal. Once mastered, this technique can be completed in two days if it is performed properly. While attempting this procedure, it is important to remember to always keep the tissue sections hydrated.After its development, this technique paved the way for researching the field of tissue regeneration to explore the autophagic process during regular and paralogical skeletal muscle regeneration in mice and human biopsies. After watching this video, you should have a good understanding of how to identify LC3 in satellite cells during muscle regeneration.

in-situ-immunofluorescent-staining-of-autophagy-in-muscle-stem-cells

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10.0K vistas.  Italian National Research Council. The overall goal of this immunofluorescent staining protocol is to monitor the autophagic process in muscle satellite cells during skeletal muscle regeneration. This method can help answer key questions in the tissue regeneration field about understanding the contribution of autophagy in deriving muscle regeneration and monitoring autophagy in satellite cells. The main advantage of this technique is that the autophagic process can be directly detected in satellite cells in tissue section from...