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

Dit werk werd ondersteund door NIAMS AR064873, Epigen Project PB. P01.001.019 / Progetto Bandiera Epigenomica IFT naar LL

Muizen werden gefokt en gehandhaafd volgens de standaard dierlijke faciliteit procedures en alle experimentele protocollen werden goedgekeurd door de Animal Welfare Assurance en het interne Animal Research Ethical Committee volgens het Italiaanse Ministerie van Volksgezondheid en voldoet aan de NIH Guide for Care and Use of Laboratoriumdieren. 1. Spierbesering en het In vivo Blok van Autofagische Flux <ol><li> Spierbesering. <ol><li> Om acuut skeletspierletsel te ...

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<html> Dit protocol beschrijft hoe autofagie in stamcellen van de skeletspier wordt gecontroleerd tijdens compenserende spierregeneratie. Verscheidene antilichamen voor het co-stainen van LC3 en MyoD werden geprobeerd, en degenen die in muisweefselafdelingen werken en succesvolle resultaten opleveren, worden hier vermeld (zie Materialtabel ). De permeabilisatie met methanol (zie stap 3.2.2) wordt sterk aanbevolen voor succesvolle kleuring. De beperking van dit protocol is gek...

Skeletspierregeneratie is het gevolg van de interactie tussen volwassen stamcellen (Spier Satellietcellen, MuSCs) en andere celsoorten die betrokken zijn bij het regeneratieve proces. Spierhomeostase en functionaliteit worden gehandhaafd door de gecombineerde signalen die voortvloeien uit de spiernis en systemische cues 1 , 2 . Gedurende het hele leven zijn veranderingen in de MuSC-functionaliteit, de spiernis en de systemische signalen aangemeld, waardoor de functionele capaciteiten bij ouderen dalen 3 . MuSCs worden in een nis onder de basale lamina geplaatst en bij spierbesering geactiveerd om beschadigde spieren 4 , 5 te repareren. Om een ​​productief regeneratief antwoord te waarborgen is het van cruciaal belang dat MuSCs verschillende processen coördineren die nodig zijn voor het verlaten van stilstand, de zelfvernieuwing en de proliferatieve expansie fase gevolgdDoor de myogene differentiatie 6 . Bij ouderen en bij spierchronische aandoeningen worden al deze functies gecompromitteerd, wat leidt tot gewijzigde spierfunctionaliteit 2 , 3 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 . Macroautophagy (hierna aangeduid als autofagie) komt tot uiting als een cruciaal biologisch proces dat essentieel is voor het behouden van weefsel homeostase 14 . Het autofagische proces omvat handelmeganismes, waarbij delen van cytoplasma, organellen en eiwitten in vesicles worden opgezwollen die uiteindelijk worden afgebroken via de lysosoomweg, het bevorderen van de verwijdering van toxische moleculen en de recycling van macromolecules. Dit biedt energie-rijke verbindingen om cel- en weefselaanpassing onder stress of andere ongunstige omstandigheden 15 , 16 te ondersteunen. Samen met de celoverlevingsactiviteit kan autofagie ook werken als een celdoodinducer, afhankelijk van de celweefselcontext ( bijv. Normaal versus kankerweefsel) en het type stressstimulus 17 , 18 . Recent bewijzen wijzen erop dat autofagie nodig is om spiermassa en myofiberintegriteit 19 , 20 te behouden en is gerapporteerd te zijn in verschillende spierdystrofieën 21 , 22 , 23 , waaronder Duchenne Spierdystrofie (DMD) 24 , 25 , 26 , 27 </suP&gt; , 28 , 29 , 30. Op dezelfde manier is een progressieve vermindering van het autofagische proces waargenomen bij ouderen 31 , 32 , 33 , 34 , 35 , na verlies van spiermassa (aangeduid als sarcopenie) 32 , 33 , 34 , 35 , 36 , 37 en in het overleven van myofiber 38 . Een nauw verband tussen autofagie en het regeneratieve potentieel van skeletspieren werd verwacht door een studie van het laboratorium van Wagers, waaruit blijkt dat een caloriebeperking de beschikbaarheid en de activiteit van MuSC verhoogt 39 . Dit nrWerd verder ondersteund door de recente observatie dat de Foxo3-Notch-as het autofagische proces activeert tijdens zelfvernieuwing 40 en de MuSC-overgang van de rustende naar de prolifererende toestand 41 . Deze gegevens komen overeen met de geleidelijke vermindering van basale autofagie van jonge tot oude en geriatrische MuSCs, in samenhang met de numerieke en functionele afname van MuSC&#39;s tijdens veroudering 42 . In een recent document bleek een nauwe relatie tussen autofagie en de compenserende spierregeneratie die de vroege stadia van DMD-progressie onderscheidt. Bijgevolg observeerden we een verminderde autofagische flux bij latere stadia van de ziekteprogressie, wanneer spierregeneratie gecompromitteerd is en fibrotische weefselafzetting optreedt. Opvallend, we hebben aangetoond dat bij regenererende omstandigheden autofagie geactiveerd wordt in MuSCs en dat het moduleren van het autofagische proces invloed heeft op MuSC-activering en fuNctionaliteit 30 . Al deze gegevens duiden de urgentie op om het autofagische proces in MuSCs te onderzoeken tijdens spierregeneratie tijdens normale en pathologische omstandigheden en gedurende de levensduur. Hier voorzien we in een protocol om het autofagische proces in MuSCs te controleren in spierregeneratieve condities door in situ immunostaining voor microtubule-geassocieerde eiwit 1A / 1B-lichte keten 3 (LC3), een marker van autofagie 43 en MyoD, een marker van Myogene afstamming, in spierweefselsecties van controle en gewonde muizen. De gemelde methode maakt het mogelijk om het autofagische proces in een specifiek celcompartiment, de MuSC, te monitoren, die een sleutelrol speelt bij het regenereren van spieren.

<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

Toenemende bewijzen wijzen op autofagie als een cruciaal regulerend proces om weefsel homeostase te behouden. Het is bekend dat autofagie betrokken is bij ontwikkeling van de skeletspier en regeneratie, en het autofagische proces is beschreven in verscheidene spierpathologieën en leeftijdsgebonden spierstoornissen. Een recent beschreven blok van het autofagische proces dat correleert met de functionele uitputting van satellietcellen tijdens spierherstel ondersteunt het idee dat actieve autofagie gekoppeld is aan productieve spierregeneratie. Deze gegevens onthullen de cruciale rol van autofagie bij satellietcelactivering tijdens spierregeneratie in zowel normale als pathologische aandoeningen, zoals spierdystrofieën. Hier bieden we een protocol om het autofagische proces in het volwassen spierstamcel (MuSC) compartiment te monitoren tijdens spierregeneratieve condities. Dit protocol beschrijft de opzetmethode om in situ immunofluorescentie beeldvorming van LC3, een a uit te voerenUtophagy marker, en MyoD, een myogene afstammingsmarkering, in spierweefselsecties van controle en gewonde muizen. De gemelde methode maakt het mogelijk om het autofagische proces in een specifiek celcompartiment, het MuSC compartiment, te monitoren, dat een centrale rol speelt bij het regenereren van spieren.

Dit protocol beschrijft een efficiënte in situ methode om autofagie in MuSCs te detecteren tijdens spierregeneratie. CTX In Vivo Behandelingen: Gebruik CTX om spierbeschadiging in de TA-spieren te veroorzaken en gebruik de ongeblokkeerde spieren als controles. Aangezien autofagie zeer dynamisch is, blokkeren de autofagische flux door IP injecties van...

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

Actieve autofagie is geassocieerd met productieve spierregeneratie, die essentieel is voor het activeren van spierstamcellen (MuSC). Hier bieden we een protocol voor de in situ detectie van LC3, een autofagie marker in MyoD-positieve MuSCs van spierweefsel secties van controle en gewonde muizen.

In situ Immunofluorescente Staining of Autophagy in Spier Stamcellen

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.

Research

JoVE Journal

Biology

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Procedures for Rat in situ Skeletal Muscle Contractile Properties

Procedures for Rat in situ Skeletal Muscle Contractile Properties

Procedures voor Rat<em&gt; In situ</em&gt; Skeletspier contractiele eigenschappen

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

Fenotypische analyse en isolatie van Murine hematopoëtische stamcellen en Lineage-geëngageerde voorouders

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

Herprogrammeren menselijke somatische cellen in geïnduceerde pluripotente stamcellen (iPSCs) Met behulp van retrovirale vector met 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

Evaluatie van de spierfunctie van de m. extensor digitorum longus<em&gt; Ex vivo</em&gt; En Voorste scheenbeenspier Spier<em&gt; In situ</em&gt; In Muizen

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

Isolatie, Cultuur en Transplantatie van Muscle satelliet-cellen

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

Efficiënte Generation humane geïnduceerde pluripotente stamcellen uit menselijke somatische cellen met 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

Profilering Individuele menselijke embryonale stamcellen door kwantitatieve 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

Nefrotoxische substantie Micro-injectie in zebravis tot acuut nierfalen Model

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

Een grafische gebruikersinterface voor software-assisted tracking van eiwitconcentratie in dynamische cellulaire protrusies

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

In situ monitoring van transitieve gevormde moleculaire Chaperone-assemblages in bacteriën, gist en menselijke cellen

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 ...