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

This study was supported by the Christian Doppler Research Foundation. We would like to thank Sabine Rauscher from the Core Facility Imaging at the Medical University of Vienna, Austria for support throughout the project. Primary antibodies were developed by Schiaffino, S., obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA.

All procedures including animal subjects were conducted in compliance with the principles of laboratory animal care as recommended by FELASA26. Approval was obtained prior to the study by the institutional review board of the Medical University of Vienna and the Austrian Ministry for Research and Science (BMWF: Bundesministerium fuer Wissenschaft und Forschung, reference number: BMWF-66.009/0222-WF/II/3b/2014).
1. Muscle Harvest
NOTE: A previous publication by Meng...

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Here, we demonstrate a widely accessible methodology to study and automatically quantify the muscle fiber populations of rat cross sections via immunohistochemistry in a time efficient manner. For reproducibility, we present a detailed step by step description and potential modifications for applications in other species not described in this study. Furthermore, we discuss the advantages of the procedure, prerequisites for optimal function and its limitations.
Currently, a number of staining m...

Skeletal muscle composition undergoes profound changes during physiological processes such as aging1,2, exercise3,4,5,6,7, or pathophysiological processes such as disease8,9,10 or trauma11. Hence, several fields of research concentrate on the structural effects of these processes to understand functional changes. One of the key aspects determining muscle function is the composition of muscle fibers. Muscle fibers express different myosin heavy chain (MHC) proteins and are thereby classified into slow, intermediate, or fast fibers7,12,13,14,15,16,17. Physiologically, muscles have different muscle fiber compositions depending on their function in the body. Using muscle fiber typing, fiber populations can be quantified to identify adaption to physiological or pathophysiological processes7,17. Historically, a number of time-consuming methods have been applied to differentiate between muscle fiber types. For this purpose, muscle fibers were classified either by reactivity of myosin ATPase at various pH levels or muscle enzyme activity. As different fiber qualities could not be assessed in a single section, multiple cross sections were required to identify all muscle fibers and allow manual quantification14,16,17,18,19,20,21,22. In contrast, recent publications used immunohistochemistry (IHC) against myosin heavy chain protein to rapidly stain multiple fiber types in a single cross sections. Based on the advantages of this procedure, it is now considered the gold standard in muscle fiber population analysis19,23,24. Using improved IHC staining protocols, we were recently able to show that the fully automatic acquisition of whole muscle cross sections and subsequent automatic muscle fiber quantification is feasible using the open source platform ImageJ. Compared to manual quantification, our procedure provided a significant decrease in time (approximately 10% of manual analyses) required per slide while being accurate to &#177;4%25.
The overall goal of this method is to describe a rapid, reliable, user-independent guide to automatic muscle fiber quantification in whole rat muscles using an open source platform. In addition, we describe potential modifications that would permit its use for other specimens such as mice or human muscles.

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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

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Whole rat muscle cross sections were stained rapidly using immunohistochemistry to identify MHC I, IIA and IIB muscle fibers. Using a fluorescent microscope slide scanner, entire cross sections were then automatically acquired for automated muscle fiber analyses with ImageJ. The concept of the procedure is based on providing a simple, reliable and time-efficient workflow for quantification of muscle fibers.
The procedure&#39;s w...

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

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

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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