Wir sind dankbar die Boettcher-Stiftung und die Amyotrophe Lateralsklerose Association (#17-II-344) für ihre Unterstützung dieser Forschung.

alle Verfahren, die Mäuse wurden von der University of Colorado-Anschutz Medical Campus institutionelle Animal Care und Use Committee genehmigt (91813(05)1D).
 1. Tag1: primäre (1°) Immunostaining mit Bovine Serum Albumin (BSA) Blockierung <ol><li>an der Luft trocknen Gefrierschnitte Maus Megalosauridae Muskel (z. B. m. Tibialis anterior, Soleus) montiert auf aufgeladene Folien für ~ 30 min 11. zeichnen Sie einen Rahmen um die Abschnitte, die mit einem Stift hydrophobe Barriere PAP.</li>
	<li>Ort ~ 250 µL 5 % BSA/Phosphat gepufferte Kochsalzlösung (BSA/PBS) auf jeder Folie, unspezifische Antikörperbindung zu blockieren. Inkubieren Sie Folien bei Raumtemperatur für 1 h</li>
	<li>Beim Blocken, bereiten 01:50 Verdünnungen des 1° Antikörpers Überstand in 5 % BSA/PBS (alle Maus monoklonale Antikörper sind; siehe Tabelle 1- 6 ,- 12) 250 µL vorbereiten Lösung pro Folie. ICE 1° Antikörper Aktien und Verdünnungen behalten</li>
</ol> <img alt="Table 1" src="/files/ftp_upload/56024/56024table1.jpg" /> Tabelle 1: Primärantikörper verwendet, um zu unterscheiden MyHCs.
<ol start="4"> <li>nach Aspiration von 5 % BSA/PBS blockierende Lösung hinzufügen 250 µL 1° Antikörper Verdünnung auf die entsprechenden Folien. Fügen Sie 250 µL 5 % BSA/PBS, sekundäre (2°) Antikörper nur negative Kontrolle gleitet.</li>
	<li>Inkubation bei 4 ° C für 24-48 h in einer feuchten Umgebung. 
	Hinweis: Eine Petrischale in Alufolie mit angefeuchtetes Filterpapier bedeckt als eine geeignete Inkubationszeit Kammer dienen können.</li>
</ol> 2. Tag 2:2 ° Immunostaining mit fluoreszierenden Antikörpern <ol><li>Aspirieren 1° Antikörperlösung oder 5 % BSA/PBS Kontrolllösung. Waschen alle Folien drei Mal 10 min mit 5 % BSA.</li>
	<li>Beim Waschen, vorbereiten 1: 200 Verdünnungen der gereinigten, Fluorophor konjugiert 2° Antikörper in 5 % BSA/PBS (siehe Tabelle 2, alle sind Ziege Anti-Maus). Bereiten Sie 250 µL Lösung pro Folie. Halten Sie 2° Fluorophor-konjugierten Antikörpern im Dunkeln auf Ice</li>
</ol> <img alt="Table 2" src="/files/ftp_upload/56024/56024table2.jpg" /> Tabelle 2: Fluorophor-konjugierten Sekundärantikörper verwendet, um zu visualisieren primären Antikörper Anerkennung von MyHCs.
<ol start="3"> <li>nach Aspiration der dritten Anwendung von 5 % BSA/PBS blockierende Lösung, 250 µL 2° Antikörper Verdünnung auf alle Folien hinzufügen. 90 min bei Raumtemperatur in einem dunklen, feuchten Umgebung inkubieren.</li>
	<li>Aspirat 2° Antikörperlösung. Waschen alle Folien drei Mal 10 min mit 5 % BSA/PBS.</li>
	<li>Spülen mit PBS. Für 10 min. trocken</li>
	<li>Montieren das Deckglas mit eine befristete, niedrigviskose wässrige Eindeckmedium.</li>
	<li>Nach dem Trocknen die Folie Schnittflächen mit Nagellack versiegeln. Trocknen und bewahren Sie in einem dunklen Slidebox.</li>
</ol> 3. Folien mit Epifluoreszenz Microscopy Imaging <ol><li>reinigen Sie die Folien mit einem kleinen, 70 % Ethanol getränkten Lab-abwischen.</li>
	<li>Erhalten digitale Bilder von Immunostained Muskel Abschnitte mit einem Epifluoreszenz-Mikroskop mit einem fotografischen Apparat (siehe die Tabelle der Materialien) ausgestattet.
	<ol><li>Wählen Sie einen Bereich von ungefähr 1 mm 2 (10 X Objektiv, 1,4 NA).</li>
		<li>Ansicht Alexa 488 konjugiert 2° Antikörper Fluoreszenz über eine 505 nm lang-Pass-Filter. Anzeigen der Fluoreszenz durch Anregung von Alexa 594 konjugiert 2° Antikörper über einen 595 nm lang-Pass-Filter erzeugt. Digitalisieren von Bildern mit einem PC-Computer mit einem kompatiblen imaging-Software ausgestattet. Gehören die Maßstabsleiste von imaging-Software für die spätere Analyse.</li>
	</ol></li></ol> 4. Analyse von Bildern mit Fidschi Hinweis: bevor Sie mit der Analyse, Fidschi (frei von den National Institutes of Health, Bethesda, MD erhältlich) über https://imagej.net/Fiji/Downloads installiert werden muss. Auch, wird das Makro in diesem Prozess verwendet in der Ergänzenden Datei bereitgestellt. Legen Sie die Makrodatei in einem leicht zugänglichen Verzeichnis.
<ol><li>Last ein Hellfeld Bild eines ausgewählten Bereichs in Fidschi. Navigieren Sie zu Plugins &#8594; Segmentierung &#8594; trainierbar Weka Segmentierung.</li>
	<li>Um die zelluläre Bereiche des Abschnitts, vorschreiben zeichnen Sie eine Linie auf einer Faser, und klicken Sie auf &#34; Klasse 1 hinzufügen &#34;. Dann zeichnen Sie eine Linie auf den Raum zwischen den Fasern, und klicken Sie auf &#34; Klasse 2 hinzufügen &#34; anlässlich der extrazellulären Domänen. Wiederholen Sie, bis in jeder Klasse gibt es 5-10 Etiketten.
	<ol><li>Klick Zug Sichter. Auf die daraus resultierenden roten und grünen Overlay mehr Beschriftungen manuell hinzufügen (siehe Punkt 4.2), nicht falsch segmentierte Stücke des Bildes, wenn nötig. Wiederholen Sie, bis die Fasern (rot) aus dem Raum zwischen den Fasern (grün) entsprechend getrennt werden.</li>
	</ol></li> <li>Klicken Sie &#34; erhalten Wahrscheinlichkeit &#34; und speichern Sie das Bild in einem neu beschrifteten Ordner.</li>
	<li>Wiederholen Sie das oben beschriebene Verfahren (Schritte 4.2 4.3) mit dem fluoreszierenden Bild vom gleichen Feld übernommen. Beschriften Immunostained Fasern als &#34; Klasse 1 &#34; und alle nicht-fluoreszierende Regionen als &#34; Klasse 2 &#34;. Speichern Sie die resultierende Wahrscheinlichkeit Karte im selben Ordner wo das verarbeitete Hellfeld-Bild gespeichert.</li>
	<li>Öffnen Sie eine der zuvor gespeicherten Wahrscheinlichkeit Karten in Fidschi. Zeichnen einer geraden Linie auf der Skala von der imaging-Software zur Verfügung gestellt. Gehe zu analysieren &#8594; Maßstab festlegen. Ändern Sie die Parameter (d.h., bekannten Abstand und Einheit der Länge) auf ihre entsprechenden Werte als von der Maßstabsleiste gegeben, dann überprüfen Sie die &#34; Global &#34; Feld, um den Maßstab für jedes Bild zu standardisieren.</li>
	<li>Navigieren zu Plugins &#8594; Makros &#8594; laufen &#8594; Tyagi Et Al. Faser Quantifizierung macro.ijm (siehe die Zusätzliche Datei). Sofort erscheint ein Navigationsbereich. Öffnen Sie das Bild &#39; s Host Ordner; ein Dialogfeld wird angezeigt. Für jedes Dialogfeld ändern Sie die &#34; Hintergrund &#34; Dropdown-Wert zu &#34; Licht. &#34; Hinweis: der Ordner wird nun mit Bildern der Faser Umrisse und Tabellenkalkulationsdateien Software der Ergebnisse (CSA und Feret Durchmesser sind am relevantesten, gefüllt Quantifizierung der Faser Morphologie; gemessenen Parameter gesetzt Messungen analysieren einstellbar). Faser Morphologie Daten generiert durch die Pore-Analysefunktion von Fidschi Tabellenkalkulation Software-Dateien für beide Leuchtstoff- und hellen Bereich Bilder automatisch übertragen werden.</li>
	<li>Berechnet den Anteil der Fasern mit dem Ausdruck einer bestimmten MyHC in einem Feld dividiert die Anzahl der Fluoreszierende Fasern im Feld durch die Anzahl der insgesamt Fasern in das entsprechende Hellfeld Bild.</li>
</ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essentiality+of+histo-+and+cytochemical+studies+of+skeletal+muscle+in+the+investigation+of+neuromuscular+disease.">The essentiality of histo- and cytochemical studies of skeletal muscle in the investigation of neuromuscular disease.</a> Neurol. 12, 778-784 (1962).</li><li>Dobrowolny, G., 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+is+a+primary+target+of+SOD1G93A-mediated+toxicity.">Skeletal muscle is a primary target of SOD1G93A-mediated toxicity.</a> Cell Metab. 8, 425-436 (2008).</li><li>Sultana, N., 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=Restricting+calcium+currents+is+required+for+correct+fiber+type+specification+in+skeletal+muscle.">Restricting calcium currents is required for correct fiber type specification in skeletal muscle.</a> Development. 143 (9), 1547-1559 (2016).</li><li>Guth, L., Samaha, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+the+histochemical+demonstration+of+actomyosin+ATPase.">Procedure for the histochemical demonstration of actomyosin ATPase.</a> Exp Neurol. 28 (2), 365-367 (1970).</li><li>Bloemberg, D., Quadrilatero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+myosin+heavy+chain+expression+in+rat,+mouse,+and+human+skeletal+muscle+using+multicolor+immunofluorescence+analysis.">Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.</a> PLoS One. 7 (4), (2012).</li><li>Lucas, C. A., Kang, L. H., Hoh, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monospecific+antibodies+against+the+three+mammalian+fast+limb+myosin+heavy+chains.">Monospecific antibodies against the three mammalian fast limb myosin heavy chains.</a> Biochem Biophys Res Commun. 272 (1), 303-308 (2000).</li><li>Lee, C. S., 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=Ca2++permeation+and/or+binding+to+Cav1.1+fine-tunes+skeletal+muscle+Ca2++signaling+to+sustain+muscle+function.">Ca2+ permeation and/or binding to Cav1.1 fine-tunes skeletal muscle Ca2+ signaling to sustain muscle function.</a> Skelet Muscle. 5 (4), (2015).</li><li>Sawano, S., 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+one-step+immunostaining+method+to+visualize+rodent+muscle+fiber+type+within+a+single+specimen.">A one-step immunostaining method to visualize rodent muscle fiber type within a single specimen.</a> PLoS One. 11 (11), (2016).</li><li>Meunier, B., Picard, B., Astruc, T., Labas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+image+analysis+tool+for+the+classification+of+muscle+fibre+type+using+immunohistochemical+staining.">Development of image analysis tool for the classification of muscle fibre type using immunohistochemical staining.</a> Histochem Cell Biol. 134 (3), 307-317 (2010).</li><li>Kammoun, M., Casser-Malek, I., Meunier, B., Picard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunohistochemical+classification+of+skeletal+muscle+fibres+in+mouse.">A simplified immunohistochemical classification of skeletal muscle fibres in mouse.</a> Eur J Histochem. 58 (2), 2254 (2014).</li><li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J Vis Exp. 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Die Autoren haben keine widerstreitenden Interessen offenzulegen.

Hier haben wir nützliche Richtung für die Identifizierung des skelettartigen Muskels Fasertypen bereitgestellt. Dabei beschreiben wir einen neuartigen Algorithmus zur Analyse der Daten.
Da unsere Ergebnisse frühere Berichte5,8,10 bestätigen weitgehend und spiegeln unsere eigenen manuellen Messungen, erscheint der Algorithmus um genau zu sein. Allerdings stießen wir auf einige seltene experimentel...

Es hat seit einiger Zeit bekannt, die Skelettmuskulatur aus Einzelfasern von vielen Arten1besteht. Zunächst wurden zwei Gruppen von Fasern anhand ihrer kontraktilen Eigenschaften charakterisiert und benannt, entsprechend langsam zuckenden (Typ I) und Fast-Twitch (Typ II). Diese Kategorien wurden auf der Grundlage der Faser Stoffwechsel weiter unterschieden. Da Typ-I-Fasern sind reich an Mitochondrien und angewiesen auf oxidativen Stoffwechsel, wurden sie aufgeklärt durch robust positiven Nicotinamid-Adenin-Dinucleotide-Tetrazolium-Reduktase (NADH-TR) Diaphorase2 oder Succinat Dehydrogenase (SDH)3 Färbung. Im Gegensatz dazu, Typ-II-Fasern weniger ausgestellt und Variablen Grad von NADH-TR Diaphorase oder SDH Färbung und wurden in zwei Fast-Twitch-Untergruppen unterteilt (Typ IIA und IIB Typ) etwas grob anhand ihrer relativen oxidativen Kapazitäten. Diese Unterscheidungen zwischen Fasern haben effektiver durch Myosin-ATPase Färbung visualisiert worden wo Typ I Fasern färben dunkel nach einer Vorinkubation bei pH 4.0 und Typ IIB Fasern absorbieren überstürzten folgende Vorinkubation bei pH 10,0 mit Typ IIA Fasern Färbung immer4.
Immunhistochemische Färbung des Myosin heavy Chain (MyHC) Isoformen hat vor kurzem eine feinere Diskriminator Fasertyp5entwickelt. Typ I, Typ IIA und Typ IIB Fasern können alle identifiziert werden mit Präzision, basierend auf ihrem MyHC Profil. Darüber hinaus eine weitere Fast-Twitch-metabolisch-Intermediate Fasertyp, Typ IIX, identifizierten6wurde. Hybrid-Fasern mit dem Ausdruck mehrere MyHC wurden auch bestätigten5,7,8. Einige Arten wie die Katze und der Pavian sind keine ausdrückliche Typ IIB MyHCs6bekannt. Obwohl MyHC Immunostaining derzeit die State-of-the-Art-Beurteilung der Muskel Zusammensetzung ist, ist die Analyse der Daten über diese Technik umständlich und zeitaufwändig ohne automatisierte Unterstützung. Zu diesem Zweck wurden eine Handvoll semi-automatischen Methoden, um diese Daten zu analysieren entwickelten5,9,10. Hier präsentieren wir Ihnen ein relativ Standardprotokoll für immunhistochemische Identifizierung der Muskelfaser-Typ5,7,8,10, zusammen mit einer teilautomatisierten Roman Algorithmus, der Analyse der Fasertyp und Faser Morphologie mit Genauigkeit beschleunigt.

<table width="974">
	<colgroup>
		<col style="width:226pt" width="301" />
		<col style="width:140pt" width="187" />
		<col style="width:172pt" width="229" />
		<col style="width:193pt" width="257" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Bovine Serum Albumin</td>
			<td class="xl65" style="width:140pt" width="187">Sigma Aldrich</td>
			<td class="xl65" style="width:172pt" width="229">A9418-100G</td>
			<td class="xl66" style="width:193pt" width="257">5% in PBS</td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl65" height="41" style="width:226pt;height:30.75pt" width="301">Hydrophobic Barrier Pap Pen</td>
			<td class="xl65" style="width:140pt" width="187">Scientific Device Laboratory</td>
			<td class="xl65" style="width:172pt" width="229">9804-02</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Microscope Slides</td>
			<td class="xl65" style="width:140pt" width="187">Globe Scientific</td>
			<td class="xl65" style="width:172pt" width="229">1358W</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Coverglass</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Scientific</td>
			<td class="xl65" style="width:172pt" width="229">12-544-E</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Immumount</td>
			<td class="xl65" style="width:140pt" width="187">Thermo Scientific</td>
			<td class="xl68" style="width:172pt" width="229">9990402</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Nail Polish</td>
			<td class="xl65" style="width:140pt" width="187">L&#39;Oreal</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl65" height="41" style="width:226pt;height:30.75pt" width="301">Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope</td>
			<td class="xl65" style="width:140pt" width="187"></td>
			<td class="xl65" style="width:172pt" width="229">Discontinued</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">SPOT RT/KE</td>
			<td class="xl65" style="width:140pt" width="187">SPOT Imaging Solutions</td>
			<td class="xl65" style="width:172pt" width="229">RT940</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Dell Optiplex</td>
			<td class="xl65" style="width:140pt" width="187"></td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">BA-F8 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgG2b; 1:50</td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">SC-71 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monclonal mouse IgG1; 1:50</td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">BF-F3 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">6H1 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgG2b</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21145</td>
			<td class="xl66">goat anti-mouse; 1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 488 anti-IgG1</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21121</td>
			<td class="xl66">goat anti-mouse;1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgGM</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21044</td>
			<td class="xl66">goat anti-mouse;1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">OCT</td>
			<td class="xl65" style="width:140pt" width="187">Sakura Finetek</td>
			<td class="xl68" style="width:172pt" width="229">4583</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">isopentane</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Scientific</td>
			<td class="xl65" style="width:172pt" width="229">O3551-4</td>
			<td class="xl66">cool with liguid nitrogen</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">PBS</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Bioreagents</td>
			<td class="xl65" style="width:172pt" width="229">BP665-1</td>
			<td class="xl66">10X, dilute to 1X</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Kim wipes</td>
			<td class="xl66">Kimberly-Clark</td>
			<td class="xl65" style="width:172pt" width="229">06-666A</td>
			<td class="xl66"></td>
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semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition

Jahrelang wurden Unterschiede zwischen skelettartigen Muskel Fasertypen am besten durch Myosin-ATPase Färbung sichtbar gemacht. Immunhistochemische Färbung des Myosin heavy Chain (MyHC) Isoformen hat vor kurzem eine feinere Diskriminator Fasertyp entwickelt. Typ I, Typ IIA, Typ IIX und Typ IIB Fasern jetzt mit Präzision, basierend auf ihrem MyHC Profil identifiziert werden können; manuelle Auswertung dieser Daten kann jedoch langsam und unten-rechts langweilig. Schnelle, präzise Bewertung der Fasertyp Zusammensetzung und Morphologie ist in dieser Hinsicht ein sehr wünschenswert Werkzeug. Hier präsentieren wir Ihnen ein Protokoll für State-of-the-Art immunhistochemische Färbung des MyHCs in Gefrierschnitte gewonnenen Maus Megalosauridae Muskel im Konzert mit einem neuartigen semi-automatischen Algorithmus, der Analyse der Fasertyp und Faser Morphologie beschleunigt. Wie erwartet, Musculus soleus angezeigt Färbung für Typ I und Typ IIA Fasern, aber nicht für Typ IIX oder Typ IIB Fasern. Auf der anderen Seite der Tibialis anterior-Muskel setzte sich überwiegend aus Typ IIX und Typ IIB Fasern, ein kleiner Bruchteil der Typ IIA Fasern und wenig oder gar keine Typ-I-Fasern. Mehrere Bildtransformationen wurden verwendet, um die Wahrscheinlichkeit Karten zum Zwecke der Messung der verschiedenen Aspekte der Faser Morphologie (d.h.Querschnittsfläche (CSA), maximale und minimale Feret Durchmesser) zu generieren. Die Werte für diese Parameter dann mit manuell erhaltenen Werten verglichen wurden. Keine signifikanten Unterschiede zwischen beiden Modi der Analyse im Hinblick auf CSA, maximal- bzw. Minimalwert Feret Durchmesser beobachtet wurden (alle p &#62; 0,05), die Genauigkeit unserer Methode angibt. So kann unser Immunostaining Analyse Protokoll zur Untersuchung der Auswirkungen auf die Muskelmasse Zusammensetzung in vielen Modellen des Alterns und Myopathie angewendet werden.

Megalosauridae Muskeln (d.h., m. Tibialis anterior und Soleus) von einer männlichen C57BL/6-Maus des unbekannten Alters seziert wurden Flash-durch das Versenken eines Kunststoff-Formenbau, enthalten die Muskeln im OAT Verbindung in flüssigem Stickstoff gekühlt Isopentane eingefroren. Dann, mit einem Cryotome, 8-10 µm Schnittserien wurden schneiden bei-20 ° C und anderen positiv geladenen Glas-Folien-12übertragen.
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Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Immunhistochemische Färbung des Myosin schwere Kette Isoformen als State-of-the-Art-Diskriminator des skelettartigen Muskels Fasertyp entstanden (d.h., Typ I, Typ IIA, Typ IIX, Typ IIB). Hier präsentieren wir Ihnen eine Färbung Protokoll sowie eine neuartige semi-automatischen Algorithmus, der erleichtert die schnelle Beurteilung der Fasertyp und Faser Morphologie.

Semi-automatische Analyse von Maus Skelettmuskulatur Morphologie und Fasertyp Zusammensetzung

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

semi-automated-analysis-mouse-skeletal-muscle-morphology-fiber-type

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.3K Aufrufe.  University of Colorado School of Medicine. Immunhistochemische Färbung des Myosin schwere Kette Isoformen als State-of-the-Art-Diskriminator des skelettartigen Muskels Fasertyp entstanden (d.h., Typ I, Typ IIA, Typ IIX, Typ IIB). Hier präsentieren wir Ihnen eine Färbung Protokoll sowie eine neuartige semi-automatischen Algorithmus, der erleichtert die schnelle Beurteilung der Fasertyp und Faser Morphologie.

Serie: Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-mechanischen Analyse von isolierten Skelettmuskel

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Forschung

Medizin

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Skeletal Muscle Gender Dimorphismus von Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Beurteilung der rechtsventrikulären Struktur und Funktion in Maus-Modell der Lungenarterie Verengung durch transthorakale Echokardiographie

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Menschlichen Skelettmuskelbiopsie Verfahren unter Verwendung der modifizierten Technik Bergström

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative Magnetresonanztomographie von Skelettmuskelerkrankung

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Mensch<em&gt; Vastus lateralis</em&gt; Skeletal Muscle Biopsy die Weil-Blakesley Conchotom Verwendung

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Induktion und Beurteilung der belastungsSkelettMuskelSchäden beim Menschen

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

Echokardiographische und histologische Untersuchung der kardiale Morphologie in der Maus

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Körperzusammensetzung und festsetzende Stoffwechselanalyse bei hohen Fett Fed Mäusen

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Quantitative Analyse der zellulären Zusammensetzung in fortgeschrittene atherosklerotische Läsionen der glatten Muskulatur Zelle Abstammung-Tracing Mäuse

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...