Name: a rapid automated protocol for muscle fiber population analysis in rat muscle cross sections using myosin heavy chain immunohistochemistry
Uploaded: 2017-03-28T13:00:00
Description: Watch this Scientific Journal Video about A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry at JoVE.com

Deze studie werd ondersteund door de christelijke Doppler Research Foundation. We willen graag bedanken Sabine Rauscher van de Core Facility Imaging aan de Medische Universiteit van Wenen, Oostenrijk voor ondersteuning van het gehele project. Primaire antilichamen werden ontwikkeld door Schiaffino, S., verkregen uit de Developmental Studies Hybridoma Bank, gecreëerd door de NICHD van de NIH en gehouden op de Universiteit van Iowa, Departement Biologie, Iowa City, IA.

Alle procedures met inbegrip van dierlijke proefpersonen uitgevoerd in overeenstemming met de principes van proefdier zorg zoals aanbevolen door FELASA 26. Goedkeuring werd voorafgaand aan het onderzoek, verkregen door de institutionele review board van de Medische Universiteit van Wenen en het Oostenrijkse ministerie van Onderzoek en Wetenschap (BMWF: Bundesministerium fuer Wissenschaft und Forschung, referentienummer: BMWF-66,009 / 0222-WF / II / 3b / 2014). 1. Muscle Harvest <p class...

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<html> Hier tonen we een algemeen toegankelijke methode te bestuderen automatisch kwantificeren spiervezel populaties ratten doorsneden door immunohistochemie in een tijd efficiënte manier. Voor reproduceerbaarheid presenteren wij een gedetailleerde stapsgewijze beschrijving en mogelijke modificaties voor toepassingen in andere soorten niet beschreven in deze studie. Verder bespreken we de voordelen van de werkwijze voorwaarden voor een optimale werking en beperkingen. Momenteel is een aanta...

Skeletspier samenstelling ondergaat diepgaande veranderingen in fysiologische processen zoals veroudering 1, 2, oefening 3, 4, 5, 6, 7 of pathofysiologische processen zoals de ziekte van 8, 9, 10 of 11 trauma. Vandaar dat een aantal onderzoeksgebieden concentreren op de structurele effecten van deze processen naar functionele veranderingen te begrijpen. Een van de belangrijkste aspecten van het bepalen van de spierfunctie is de samenstelling van de spiervezels. Spiervezels express zware keten (MHC) eiwitten verschillende myosine en worden daardoor geclassificeerd in langzame, tussengelegen of snelle vezels 7, 12, 13 </sup<html> &gt;, 14, 15, 16, 17. Fysiologisch spieren verschillende spiervezels samenstelling afhankelijk van hun functie in het lichaam. Gebruik spiervezels typen, kan vezelpopulaties worden gekwantificeerd aanpassing identificatie van fysiologische of pathofysiologische processen 7, 17. Historisch gezien een aantal tijdrovende methoden toegepast om onderscheid te maken tussen types spiervezels. Hiertoe werden spiervezels geclassificeerd hetzij reactiviteit van myosine ATPase bij verschillende pH-niveaus of spier enzymactiviteit. Aangezien verschillende vezelkwaliteiten niet een enkel kon worden vastgesteld, werden meerdere doorsneden nodig om alle spiervezels te identificeren en toe handmatig kwantificering 14, 16, 17,= &quot;xref&quot;&gt; 18, 19, 20, 21, 22. Daarentegen recente publicaties gebruikt immunohistochemie (IHC) tegen myosine zware keten eiwit snel vlek meerdere soorten vezels in één dwarsdoorsneden. Op basis van de voordelen van deze procedure, wordt nu beschouwd als de gouden standaard in de spiervezels populatie-analyse 19, 23, 24. Middels verbeterde IHC kleuringsprotocollen, waren we recent aantonen dat de volledig automatische verwerving van volledige spieren dwarsdoorsneden en daaropvolgende automatische spiervezels kwantificering mogelijk van de open source platform ImageJ. Vergeleken met handmatige kwantificering onze procedure een significant kortere periode (circa 10% van handmatige analyses) nodig per slede terwijl nauwkeurigheid van ± 4% 25 </sup&gt;. Het algemene doel van deze werkwijze is een snelle, betrouwbare, gebruikersonafhankelijke lijst automatisch spiervezel te kwantificeren geheel rat spieren beschrijven het gebruik van een open source platform. Daarnaast beschrijven we mogelijke modificaties dat het gebruik van andere monsters zoals muizen of menselijke spieren zou toelaten.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">O.C.T compound</td>
			<td style="width:397px;">Tissue-Tek, Sakura, Netherlands</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For embedding of muscle tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopentane</td>
			<td></td>
			<td></td>
			<td>for adequate freezing of muscle tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Ultra Plus slides</td>
			<td>Thermo Scientific, Germany</td>
			<td align="right">1014356190</td>
			<td>adhesive slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate buffered saline&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Thermo Scientific, Germany</td>
			<td align="right">85112</td>
			<td>Detergent Soluation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum</td>
			<td>Thermo Scientific, Germany</td>
			<td>50197Z</td>
			<td>Goat Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAKO Fluorescent Mounting Medium</td>
			<td>Dako Denmark</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dako pen</td>
			<td>Dako Denmark</td>
			<td>S200230-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TissueFAXSi plus&#160;</td>
			<td>TissueGnostics, Vienna, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-I (Cat# BA-F8, RRID: AB_10572253)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td>
			<td></td>
			<td>Supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-IIa (Cat# SC-71, RRID: AB_2147165)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td>
			<td></td>
			<td>Supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MHC-IIb (Cat# BF-F3, RRID: AB_2266724)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td>
			<td></td>
			<td>Supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa&#160;Fluor&#160;633&#160;Goat Anti-Mouse IgG2b&#160;</td>
			<td>Thermo Scientific, Germany</td>
			<td>A-21146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG1&#160;(&#947;1)</td>
			<td>Thermo Scientific, Germany</td>
			<td>A-21121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 Goat Anti-Mouse IgM (&#181; chain),</td>
			<td>Thermo Scientific, Germany</td>
			<td>A-21426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucBlue Fixed Cell ReadyProbes Reagent</td>
			<td>Thermo Scientific, Germany</td>
			<td>R37606</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid automated protocol for muscle fiber population analysis in rat muscle cross sections using myosin heavy chain immunohistochemistry

Kwantificatie van spiervezel populaties verschaft meer inzicht in de gevolgen van de ziekte, trauma en verschillende andere invloeden op skeletspier samenstelling. Diverse tijdrovende methoden zijn van oudsher gebruikt om vezelpopulaties studeren op vele terreinen van het onderzoek. Echter, recent immunohistochemische methoden op basis van myosine zware keten eiwitexpressie ontwikkeld te snel alternatief voor meerdere vezeltypes is uit een enkele sectie. Hier presenteren we een snelle, betrouwbare en reproduceerbare protocol voor betere kleuring kwaliteit, waardoor automatisch ophalen van hele dwarsdoorsneden en automatische kwantificatie van vezelpopulaties met ImageJ. Hiertoe zijn ingebed skeletspieren gesneden dwarsdoorsneden, gekleurd met myosine zware ketens kunnen antilichamen met fluorescente secundaire antilichamen en DAPI kleuring van celkernen. Hele dwarsdoorsneden automatisch gescand met diascanner hoge resolutie composiet te verkrijgenfoto&#39;s van het gehele monster. Vezels populatie analyses worden vervolgens uitgevoerd om langzame, intermediaire en snelle vezels behulp van een geautomatiseerde macro voor ImageJ kwantificeren. We hebben eerder aangetoond dat deze methode vezelpopulaties betrouwbare wijze identificeren mate van ± 4%. Daarnaast is deze methode minder inter-user variabiliteit en tijd per analyses aanzienlijk van de open source platform ImageJ.

Hele rattenspier doorsneden werden gekleurd met immunohistochemie snel identificeren MHC I, IIA en IIB spiervezels. Met behulp van een fluorescentiemicroscoop diascanner, werden gehele doorsneden automatisch verkregen geautomatiseerde spiervezels analyses ImageJ. Het concept van de procedure is gebaseerd op een eenvoudige, betrouwbare en tijdbesparende workflow voor het kwantificeren van spiervezels. Werkstroom van de procedur...

Watch this Scientific Journal Video about A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry at JoVE.com

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Hier presenteren we een protocol voor snelle spiervezels analyses, welke verbeterde kleuring kwaliteit en daardoor automatisch ophalen en kwantificering van vezelpopulaties door de vrij beschikbare software ImageJ mogelijk maakt.

Een Rapid Automated Protocol voor Muscle Fiber Population Analysis in Rat Muscle Dwarsdoorsneden Met behulp van zware keten van myosine Immunohistochemie

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

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Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generatie Myospheres Van hESCs door Epigenetische herprogrammeren

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Optische cross-sectionele spier gebied bepaling van Drosophila Melanogaster volwassen indirecte vlucht spieren

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Sequentiële immunofluorescentie en immunohistochemie op Gecryode zebravis-embryo's

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Bereiding van ratskeletspieren homogenaten voor nitraat- en nitrietmetingen

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Een eenvoudig en goedkoop loopwielmodel voor progressieve weerstandstraining bij muizen

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration (Video) | JoVE 

Op decellularisatie gebaseerde kwantificering van vetinfiltratie van skeletspieren

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Confocal Microscopic Analysis of Rat Muscle Mitochondria

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

Mitochondrial Remodeling in Skeletal Muscle (Video) | JoVE

Analyse van de mitochondriale dichtheid en longitudinale verdeling in levende skeletspiervezels van ratten door confocale microscopie

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of ...