Name: identification of skeletal muscle satellite cells by immunofluorescence with pax7 and laminin antibodies
Uploaded: 2018-04-19T13:00:00
Description: Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

Vi takker NIAMS Light Imaging sektion for at give mikroskoper og teknisk hjælp. MF20 og Pax7 antistofferne blev indhentet fra de udviklingsmæssige undersøgelser hybridom Bank udviklet i regi af NICHD og vedligeholdes af Department of Biological Sciences, The University of Iowa, Iowa City. Dette arbejde blev støttet af murene forskning Program af NIAMS af National Institutes of Health.

I denne protokol, blev forreste hind lemmer muskler af voksen mus (2-6 måneder), tibialis anterior (TA) og extensor digitorum longus (EDL), ansat som et eksempel til at udføre immunofluorescens farvning på deres SCs. Alle de skridt, håndtering af mus og muskel væv dissektioner er blevet godkendt af Animal Care og brug udvalg (ACUC) af NIAMS/NIH.
1. dissekere TA/EDL muskel fra mus Hind lemmer
<ol><li>Aflive mus i en CO2 fyldt eutanasi kammer under retningslinjer for ACUC af NI...

<ol>
	<li>Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+and+cell+lineages+that+underlie+the+formation+of+skeletal+muscle.">Gene regulatory networks and cell lineages that underlie the formation of skeletal muscle.</a> Proc Natl Acad Sci U S A. 114 (23), 5830-5837 (2017).</li><li>Buckingham, M., Relaix, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX3+and+PAX7+as+upstream+regulators+of+myogenesis.">PAX3 and PAX7 as upstream regulators of myogenesis.</a> Semin Cell Dev Biol. 44, 115-125 (2015).</li><li>Relaix, F., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+insect+eye+to+vertebrate+muscle:+redeployment+of+a+regulatory+network.">From insect eye to vertebrate muscle: redeployment of a regulatory network.</a> Genes Dev. 13 (24), 3171-3178 (1999).</li><li>Chang, N. C., Chevalier, F. P., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+Cells+in+Muscular+Dystrophy+-+Lost+in+Polarity.">Satellite Cells in Muscular Dystrophy - Lost in Polarity.</a> Trends Mol Med. 22 (6), 479-496 (2016).</li><li>Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cell+of+skeletal+muscle+fibers.">Satellite cell of skeletal muscle fibers.</a> J Biophys Biochem Cytol. 9, 493-495 (1961).</li><li>Kuang, S., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+biology+of+satellite+cells+and+their+therapeutic+potential.">The emerging biology of satellite cells and their therapeutic potential.</a> Trends Mol Med. 14 (2), 82-91 (2008).</li><li>Wang, Y. X., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+the+engines+of+muscle+repair.">Satellite cells, the engines of muscle repair.</a> Nat Rev Mol Cell Biol. 13 (2), 127-133 (2011).</li><li>Rinaldi, F., Perlingeiro, R. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+acts+directly+on+the+zebrafish+dermomyotome+to+promote+myogenic+differentiation.">Hedgehog acts directly on the zebrafish dermomyotome to promote myogenic differentiation.</a> Dev Biol. 300 (2), 736-746 (2006).</li><li>Juan, A. H., 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=Polycomb+EZH2+controls+self-renewal+and+safeguards+the+transcriptional+identity+of+skeletal+muscle+stem+cells.">Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.</a> Genes Dev. 25 (8), 789-794 (2011).</li><li>Jackson, K. A., Snyder, D. S., Goodell, M. 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Den ovennævnte protokol var baseret på en metode til Pax7/MF20 farvning på zebrafisk skeletmuskulatur12. Løsningerne anvendes og blokerer trin er identiske eller lignende. Antistofferne anvendes er identiske. De justerede trin var baseret på funktionerne i musen muskelvæv og SCs. Først, Laminin antistof blev tilføjet i blanding til at visualisere og bekræfte placeringen af SCs. Det var især nyttigt at tælle antallet af SCs under mikroskop, når du bruger dobbelt filter terning af 488/55...

Skeletmuskulatur er sammensat af multinucleated muskelceller, kaldet myotubes, organiseret i myofibers, som genererer kraft og bevægelser gennem sammentrækning. Mest skeletmuskulatur, bortset fra nogle kraniofaciale muskler, er afledt af en midlertidig embryonale struktur kaldet en somite1. Myogenic forløber celler delaminere fra den epitel somite at blive myoblasts. Myoblasts yderligere differentiere i myocytes, der smelter sammen og bliver myotubes til at danne multi nucleated myofibers. Ovenstående proces kaldes myogenesis og er kendetegnet ved tidsligt reguleret kontrol af genekspression. Myogenic prækursorer express Pax3 og Pax7, mens myoblasts express MyoD og/eller Myf5 og myocytes express myogenin og myosins2,3. Muskelvækst er en proces, hvor myofibers bliver større ved at integrere flere myonuclei i eksisterende fibre (hyperplasi) og af en stigning i muskel fiber størrelse (hypertrofi)4. Under muskelvækst er der en bæredygtig kilde til myogenic celler, der har stamcelleforskning egenskaber i, at de kan skelne og selv forny. Disse celler kaldes satellit celler baseret på deres fysiske placering mellem sarcolemma (celle membran af myofiber) og basal lamina5. SCs energisk bidrage til muskelvækst i juvenil fase (de første 2-3 uger af postnatal mus), men bliver inaktiv i hvile voksen muskel6. Bemærkelsesværdigt, de kan aktiveres igen i svar til muskel skader og differentiere sig til nye muskelceller til at reparere beskadigede muskel7.
Stamcelle egenskaber gør studiet af SCs relevante for både grundlæggende muskelbiologi og terapier af muskel sygdomme8. Som et resultat, har det været et område med intens undersøgelse i de seneste årtier. Er sket en enorm fremskridt i dissekere genetik og Epigenetik SCs9,10. Teknikker involveret i isolering og identifikation af SCs i situ var udviklet og optimeret langs vej11. Immunfluorescent farvning tillader identifikation af SCs ved brug af specifikke antistoffer, herunder for Pax7. Dog render knaphed og lille størrelse af SCs kombineret med en stærk auto-fluorescens af voksne skeletmuskulatur væv visualisering udfordrende. Her, vi beskriver en immunfluorescent farvning protokol optimeret til musen muskelvæv for Pax7 og baseret på en eksisterende metode for zebrafisk muskel12. Derudover er en Laminin antistof mærket med en særskilt fluorophore ansat til at identificere basal lamina hvorunder SCs er placeret. Denne protokol tillader konsekvent visualisering af Pax7-positive SCs og myogenic prækursorer under alle testede fysiologiske forhold og udviklingsstadier.

<table>
	<tbody>
		<tr>
			<td>methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>M32631</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature (O.C.T.) compound</td>
			<td>Electron Microscopy Sciences</td>
			<td>62550-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco, Themo Fisher</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>16% PFA</td>
			<td>TED PELLA</td>
			<td>50-00-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Thermo Fisher</td>
			<td>0 1-6201</td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td>115-007-003</td>
			<td></td>
		</tr>
		<tr>
			<td>20x Citrate Buffer</td>
			<td>Thermo Fisher</td>
			<td>00 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pax7 mono-clonal mouse antibody (IgG1) (supernatant)</td>
			<td>Developmental Study Hybridoma Bank</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin polyclonal rabbit antibody</td>
			<td>Sigma-Aldrich</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr>
			<td>MF20 mono-clonal mouse antibody (IgG2b) (supernatant)</td>
			<td>Developmental Study Hybridoma Bank</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A-21121</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG2b cross-absorbed secondary antibody, Alexa Fluor 647</td>
			<td>Thermo Fisher</td>
			<td>A-21242</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555</td>
			<td>Thermo Fisher</td>
			<td>A32732</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1860 cryostat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DM6000 wide-field fluorescent microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMR wide-field fluorescent microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM510 confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM780 confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cuisinart electronic pressure cooker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of skeletal muscle satellite cells by immunofluorescence with pax7 and laminin antibodies

Immunfluorescens er en effektiv metode, der hjælper til at identificere forskellige celletyper på væv sektioner. For at studere befolkningens ønskede celle, anvendes antistoffer for bestemte celle markører på væv sektioner. I voksne skeletmuskulatur er satellit celler (SCs) stamceller, der bidrager til muskel reparation og regeneration. Det er derfor vigtigt at visualisere og spore satellit cellen befolkningen under forskellige fysiologiske tilstande. I hvile skeletmuskulatur, bor SCs mellem basal lamina og myofiber plasmamembran. Et almindeligt anvendte markør til at identificere SCs på myofibers eller i cellekultur er parrede boks protein Pax7. I denne artikel præsenteres en optimeret Pax7 immunofluorescens protokol på skeletmuskulatur sektioner der minimerer uspecifik farvning og baggrund. Et andet antistof, der genkender et protein (laminin) af basal lamina blev også tilføjet for at hjælpe med at identificere SCs. lignende protokoller kan også bruges til at udføre dobbelt eller tredobbelt mærkning med Pax7 og antistoffer for ekstra proteiner af interesse.

Efter ovenstående trin, kan SCs visualiseres med succes i voksen hvilende muskel sektioner under en fluorescerende mikroskop (figur 1). Selv om den voksne muskelvæv har stærke auto-fluorescens i visse typer af fibre, de lyse Alexa serien farvestoffer kan overvinde baggrundsstøjen og signalet skiller sig ud (figur 1A, B, pilespidser). To-foton Konfokal mikroskopi indfanger et relativt renere billede (<strong c...

Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

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

Nøjagtig identifikation af satellit celler er afgørende for at studere deres funktioner under forskellige fysiologiske og patologiske betingelser. Denne artikel præsenterer en protokol for at identificere satellit celler på voksne skeletmuskulatur sektioner af immunofluorescens-baserede farvning.

Identifikation af skeletmuskulatur satellit celler ved immunfluorescens med Pax7 og Laminin antistoffer

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

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