We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1. Tissue Ernte und Einfrieren von Muskelgewebe <ol><li> Euthanize die Maus mit einer Überdosis Isofluran (2-Chlor-2- (difluormethoxy) -1,1,1-trifluor-ethan). Führen Sie diesen Schritt in einem Abzug und verwenden Sie eine Auffangverfahren, wie einem Exsikkator oder einer Glasglocke, die ein Wattestäbchen in Isofluran getränkt. </li><li> Tod Bestätigen fest drückte Fußballen. Verwenden Sie eine sekundäre Methode der Euthanasie wie Genickbruch. </li><li> Befeuchten Sie das Fell mit 70% Alkohol, um das Anhaften von losen Haarsträhnen auf die Muskeln zu reduzieren. Schälen Sie die Haut von dem Schenkel mit einer feinen Pinzette, wie # 5. </li><li> Trennen Sie vorsichtig die Muskeln von Interesse (tibialis anterior (TA) in diesem Fall) von den Knochen unter, ausgehend von der Sehne und zu entfernen. </li><li> Auszuschneiden den Muskel, bedecken Sie es in OCT (optimale Schnitt Temperatur-Verbindung), und legen Sie ihn flach auf einem markierten Einweggefrierform. Stellen Sie sicher, dass der Muskel an seinem normalen physiologischen Orientierung (Kopf bis tail) ohne Dehnung. Alternativ einfrieren den Muskel auf kleinen Styropor-Plätze und stecken Sie es mit Insektenstifte auf die richtige Länge der Muskeln zu gewährleisten. </li><li> Chill-2-Methylbutan (Isopentan) in einem Stahlbecher mit Hilfe von flüssigem Stickstoff. Dies dauert im Durchschnitt 3-5 min. Halten Sie das Rühren mit Unterbrechungen bis weiße Niederschläge beginnen, sich auf den Boden und die Wände des Bechers zu bilden. HINWEIS: Wenn dies geschieht, Isopentan optimale Gefriertemperatur erreicht hat (150 ° C) </li><li> Tauchen Die Formen, die Muskeln in der gekühlten 2-Methylbutan. Einfrieren kleinen Muskeln, wie der Membran und extensor digitorum longus (EDL) für 6-12 sec und größere Muskeln wie gastrocnemius soleus (GS) und Trizeps für 15-20 sec. Die Muskeln Nicht einfrieren zu lange, da dies zu Rissen führen. </li><li> Übertragen Sie die gefrorenen Gewebe auf Trockeneis und lassen Sie die Isopentan verdampft (beachten Sie, dass dieser Prozess dauert im Durchschnitt etwa 15 bis 20 min). Wickeln Sie das Gewebe in Aluminiumfolie und im Kühlschrank bei ultra niedriger Temperatur (-70 ° C bis -80 ° C) bis zum Schneiden. </li></ol> 2. Einbindung der Gewebe in OCT zu Tissue-Block vorbereiten <ol><li> Transport Gewebe auf Trockeneis zu cyrostat zu Auftauen zu verhindern. Transfer Gewebe Kryostatkammer (bei -20 bis -24 ° C eingestellt, um volle Auftauen der Gewebe zu vermeiden) und lassen Sie sie für 30 Minuten ins Gleichgewicht kommen. </li><li> Drehen Sie das Peltier-Kühlschacht auf. Wenn der Kryostat nicht diese Kühlung haben, verwenden Aerosolkühlspray, schnell einfrieren Oktober Lassen Sie sich nicht den Muskel Tauwetter zu irgendeinem Zeitpunkt während der Einbettung, da dies zu Frostschäden führen. </li><li> Fügen Sie eine gleichmäßige dünne Schicht von Oktober auf der Probenscheibe und abkühlen lassen. Wenn die meisten der Oktober ist auf dem Boden, während immer noch Flüssigkeit auf der Oberseite eingefroren, legen Sie die Gewebe an der richtigen Ausrichtung (senkrecht zur OCT zur Querabschnitte und parallel Oktober für Längsschnitte). Benutzen Sie Aerosol-Kühlspray, schnell einfrieren Oktober Tippen Sie auf die unembeded Teil des Gewebes mit Wärme extRactor und warten Sie 45 Sekunden. </li><li> Fügen Sie eine weitere dünne Schicht Oktober rund um die Muskeln, Aerosolspray mit Kühlspray und extrahieren Sie die Hitze wie im vorherigen Schritt. </li><li> Halten Sie das Hinzufügen Oktober in kleinen Schritten und schnell einfrieren des OAT mit einer Kombination von Aerosol-Kühlspray und Wärmeableitblock bis der ganze Muskel wird abgedeckt. </li><li> Legen Sie die Wärmeableitblock auf der Oberseite der Gewebeblock, und warten Sie 5 Minuten. </li></ol> 3. Vorbereitung Profile und Identifizieren der Teile von Mid-Bauch Region der Muskeln <ol><li> Montieren Sie die Probe auf die Probe Kopf. Ziehen Sie den Knopf, um die Disc zu sichern. Stellen Sie sicher, dass die Objektplatte ist in einem guten Kontakt mit dem Objektkopf. </li><li> Passen Sie die Raumtemperatur zwischen -21 und -24 ° C, und warten Sie, bis die eingestellte Temperatur erreicht ist. </li><li> Passen Sie die Einstellungen Dicke (7 &amp; mgr; m); schneiden Sie den Block durch Vorschieben oder Zurückziehen des Blocks mit Grob- und Feineinstellungen. </li><li> Sammeln Sie die AbschnitteAuf vorgewärmten positiv geladenen Objektträgern durch Drücken der Oberseite des Schiebers auf die Abschnitte mit der Anziehung zwischen entgegengesetzten Ladungen, um die Haftung der ausgeschnittenen Abschnitte zu der Folie zu erleichtern. </li><li> Identifizieren Sie die Mitte der Bauchabschnitte durch Messung Querschnittsfläche (CSA) der Abschnitte. Tun Sie dies mit Imaging-Software auf dem Computer, an das Mikroskop angeschlossen ist. HINWEIS: Phasenkontrastbilder aufgenommen und Umfang des gesamten Muskel wird über eine Rückverfolgung-Funktion auf dem Software-Programm gemessen. Dies wird sowohl CSA sowie minimale Frettchen Messer (Maß für die Faserquerschnittsgröße, die als der kleinste Abstand zwischen zwei parallelen Tangenten an gegenüberliegenden Seiten eines Teilchens ausgedrückt wird) Messungen zu erhalten. Hinweis: Bei der CSA und Frettchen Mindestdurchmessermessungen beginnen, Plateau, hat Mitte Bauch erreicht ist. </li><li> Inventur und Archivierung der Objektträger bei -80 ° C für die künftige Färbung. HINWEIS: Oktober muss nicht vor der entfernt werden, um staining Verfahren. Sobald Folien eingedeckt werden, sind sie bereit, sofort gefärbt werden. </li></ol> 4. Die Färbung HINWEIS: Während ausführliche Schritt-für-Schritt-Verfahren finden Sie auf der Produkt-Datenblatt entnommen werden und sollte für Färbeverfahren befolgt werden, können persönliche Optimierung erforderlich, um eine optimale Färbung zu erhalten. <ol><li> Da die Montage Medien (siehe Tabelle) ist nicht mit Wasser mischbar ist, dehydrieren gleitet durch zunehmende Grade von Alkohol (2 min jeweils in 70%, 95% und 100% Ethanol), und deaktivieren Sie sie mit Xylol (100% für 5 Minuten), um sicherzustellen, Dias nicht undurchsichtig über die Zeit. </li><li> Deckglas Sektionen und trocknen lassen. Bild mit Mikroskop mit Bildbearbeitungssoftware ausgestattet. </li></ol>

<ol>
	<li>Bonnemann, C. 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=Diagnostic+approach+to+the+congenital+muscular+dystrophies.">Diagnostic approach to the congenital muscular dystrophies.</a> Neuromuscular disorders : NMD. 24, 289-311 (2014).</li><li>Caughey, J. E., Pachomov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diaphragm+in+dystrophia+myotonica.">The diaphragm in dystrophia myotonica.</a> J Neurol Neurosurg Psychiatry. 22, 311-313 (1959).</li><li>Fetterman, G. H., Wratney, M. J., Donaldson, J. S., Danowski, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophy.+I.+History,+clinical+status,+muscle+strength,+and+biopsy+findings.">Muscular dystrophy. I. History, clinical status, muscle strength, and biopsy findings.</a> AMA J Dis Child. 91, 326-338 (1956).</li><li>Platzer, A. C., Chase, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+Alterations+in+Preclinical+Mouse+Muscular+Dystrophy.">Histologic Alterations in Preclinical Mouse Muscular Dystrophy.</a> The American journal of pathology. 44, 931-946 (1964).</li><li>Mercuri, E., 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=Muscle+magnetic+resonance+imaging+involvement+in+muscular+dystrophies+with+rigidity+of+the+spine.">Muscle magnetic resonance imaging involvement in muscular dystrophies with rigidity of the spine.</a> Ann Neurol. 67, 201-208 (2010).</li><li>Tasca, 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=Different+molecular+signatures+in+magnetic+resonance+imaging-staged+facioscapulohumeral+muscular+dystrophy+muscles.">Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles.</a> PloS one. 7, e38779 (2012).</li><li>Girgenrath, M., Dominov, J. A., Kostek, C. A., Miller, J. B. <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+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy.">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li><li>Kumar, A., Yamauchi, J., Girgenrath, T., 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=Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice,+a+model+for+congenital+muscular+dystrophy+type+1A.">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li><li>Mehuron, 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=Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy.">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14 (2014).</li><li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., 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=Triggering+regeneration+and+tackling+apoptosis:+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A.">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li><li>Sheriffs, I. N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy.">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33, 845-853 (1985).</li><li>Maccarty, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Diagnostic+Reliability+of+Frozen+Sections.">The Diagnostic Reliability of Frozen Sections.</a> The American journal of pathology. 5, 377-380 (1929).</li><li>Shi, S. R., 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=Evaluation+of+the+value+of+frozen+tissue+section+used+as+'gold+standard'+for+immunohistochemistry.">Evaluation of the value of frozen tissue section used as 'gold standard' for immunohistochemistry.</a> Am J Clin Pathol. 129, 358-366 (2008).</li><li>Turan, 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=Accuracy+of+frozen+sections+for+intraoperative+diagnosis+of+complex+atypical+endometrial+hyperplasia.">Accuracy of frozen sections for intraoperative diagnosis of complex atypical endometrial hyperplasia.</a> Asian Pacific journal of cancer prevention : APJCP. 13, 1953-1956 (2012).</li></ol>

The authors have nothing to disclose.

Histologie und Immunhistochemie wurden als wichtige Instrumente für das Verständnis der Manifestation und Progression der Pathologie in verschiedenen neuromuskulären Erkrankungen eingesetzt. Klassischerweise wurden Muskelbiopsien ersten in Formaldehyd fixiert und dann mit Paraffinwachs eingebettet. Während Formaldehydfixierung bewahrt Gewebearchitektur und ermöglicht Gewebe Lagerung und Transport bei RT, inaktiviert es auch Enzyme und Proteine ​​Vernetzungen erfordern weitere Schritte, um genaue Immunfärbung z...

Prüfung der Muskel Histologie spielt eine entscheidende Rolle für das Verständnis der verschiedenen Arten von neuromuskulären Störungen 1-4. Wenn sie mit anderen Techniken kombiniert, erlaubt es Forschern, eine bessere Vorstellung von der Manifestation und Progression der Pathologie zu bekommen und kann auch wichtig sein, um die Wirksamkeit eines therapeutischen Interventions 5-10 bewerten. Jedoch genau zu sein, müssen die Gewebe in der richtigen Art und Weise gespeichert, die den physiologischen Zustand unmittelbar vor dem Herausschneiden erhalten. Dies erfordert große Sorgfalt bei der Entnahme, Konservierung, Schnitte, und Färbung. Gewebe können auf zwei verschiedenen Wegen für lichtmikroskopische Untersuchung vorbereitet werden. Das erste Verfahren, Formalin fixierten und in Paraffin eingebetteten (FFPE) Abschnitten erfordert Gewebe in 10% gepuffertem Formaldehyd natürlichen bevor sie mit Paraffin eingebettet 11,12 fixiert werden. Der Fixierungsschritt hilft, Morphologie besser zu bewahren, sondern auch Querverbindungen endogenen proteins macht es erforderlich komplizierte Antigen-Retrieval-Verfahren für die Immunfärbung und die Beseitigung der Möglichkeit der enzymatischen Flecken auf solche Abschnitte 12. Gefrierschnitte andererseits brauchen nicht vor, eine feste oder verarbeitet werden sollen; daher sind Proteine ​​in der Lage, native biologische Konformationen 13,14 beibehalten. Ferner gefrorene Schnitte auch schnelle Durchlaufzeit, die manchmal notwendig ist, beispielsweise in der intraoperativen Diagnose von Sarkomen und anderen chirurgischen Biopsien 15 ermöglichen. , Wenn nicht richtig gemacht, Diese Methode ist jedoch anfällig für Schäden, die Änderungen an Muskel-Architektur macht die Abschnitte ungeeignet für die histologische Untersuchung stellt einfrieren. Dieses Papier wird die effektivste Methode, um Muskelbiopsien, um eine optimale Färbung für die ordnungsgemäße Prüfung zu erreichen vorbereiten zu skizzieren.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Olympus</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Nikon Imaging Software Basic Research</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>M 32631-SL</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
	</tbody>
</table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

Histologische Bewertung von Muskelbiopsien wurde als unverzichtbares Instrument für das Verständnis der Entwicklung und Progression von Pathologie der neuromuskulären Erkrankungen serviert. Um dies zu tun, muss jedoch die richtige Pflege, wenn das Ausschneiden und die Erhaltung Gewebe, um eine optimale Färbung erreichen ergriffen werden. Eine Methode der Gewebekonservierung beinhaltet Befestigungs Gewebe in Formaldehyd und dann Einbettung mit Paraffinwachs. Dieses Verfahren bewahrt Morphologie gut und ermöglicht langfristige Lagerung bei Raumtemperatur, ist aber umständlich und erfordert Umgang mit giftigen Chemikalien. Ferner Formaldehydfixierung ergibt Antigen Vernetzung, die Antigen-Retrieval-Protokolle für eine wirksame Immunfärbung erfordert. Im Gegenteil, ist gefrorene Schnitte nicht Fixierung erfordern und somit behält biologischen Antigen-Konformation. Dieses Verfahren stellt auch einen entscheidenden Vorteil in der schnellen Durchlaufzeit, so dass es besonders hilfreich in Situationen, brauchen schnelle histologische Auswertung wie intraoperative chirurgischen Biopsien. Hier beschreiben wir die effektivste Methode zur Herstellung von Muskelbiopsien zur Visualisierung mit unterschiedlichen histologischen und immunologischen Flecken.

Das Set-up für die Ernte und das Einfrieren von Gewebe kann in 1 gesehen werden kann. Entfernte Gewebe sollte auf eine Gaze in PBS eingeweicht, um das Trocknen (1A) zu vermeiden, gespeichert werden. Werden die Gewebe so ausgewählt sind, für die histologische Analyse verwendet werden, sollten in OCT eingetaucht werden und auf Kryo Form gelegt in der richtigen Orientierung und so nahe physiologischen Länge möglichst genaue Morphologie (1B) zu gewährleisten. Aufschl?...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Gebote und Verbote in der Herstellung von Muscle Kryoschnitte für die histologische Analyse

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

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31.7K Aufrufe.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Serie: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

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 ...

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Medizin

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Hochdurchsatz-Durchflusszytometrie zellbasierten Assay auf Antikörper gegen N-Methyl-D-Aspartat-Rezeptor oder Dopamin-2-Rezeptor im menschlichen Serum Detect

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Sammlung und Extraktion von DNA-Speichel für Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Halsschlagader Infusionen für pharmakokinetischen und pharmakodynamischen Analyse von Taxanen in Mäusen

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Ultraschall Basierend Beurteilung der koronaren Herzfluss und koronare Flussreserve Mit der Drucküberlastung Modell an der Maus

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

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 ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Die Verkalkung der glatten Gefäßmuskelzellen und Imaging von Aortic Kalzifizierung und Entzündung

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissektion der Maus Bauchspeicheldrüse für histologische Analyse und Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

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 ...