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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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" width="986">
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	</colgroup>
	<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 ...

The overall goal of this method is to rapidly and reliably guide muscle fiber quantification in whole rat muscles in an automatic and user-independent manner using an open-source platform. This method can help answer key questions in the neuromuscular field about defects of aging, disease, or trauma on skeletal muscles. The main advantage of this technique is that it can be used to reliably analyze muscle fiber populations and to generate high-quality images of muscle cross-sections.After sufficient thawing, carefully rinse the specimens in PBS supplemented with Triton X, or PBST, for one 10-and one five-minute wash. After the second wash, air-dry the sections for two minutes. Then, use a hydrophobic pen to encircle the individual cross-sections.After another 15 minutes of air-drying, block any nonspecific binding with PBST supplemented with goat serum for one hour at room temperature. Next, wash the slides and add the appropriate primary antibody cocktail to each sample for a one-hour staining at room temperature protected from light. At the end of the incubation, rinse the slides with one 10-and one five-minute PBST wash.Then, label the muscle sections with the appropriate secondary antibody cocktail for one hour protected from light. After washing the slides two times in PBST, as demonstrated, dry the slides off and then stain the nuclei with a DAPI nuclear staining kit according to the manufacturer's instructions. Remove the excess DAPI with a one-minute PBST wash, followed by their brief air-drying.Then, cover the samples with fluorescent mounting medium and cover slips, and store the slides at four degrees Celsius protected from light. Within 24 to 48 hours of staining, load the slides into a slide scanner and open a new project in the program software. For the slide preview, select the DAPI channel and the 2.5x lens, and manually focus on the first specimen.Outline each cross-section that should be included for the detailed acquisition process and set the exposure times for each channel. Now, run the automatic image acquisition, checking for the correct acquisition of the first field of view, to prevent an incorrect acquisition early in the automated process. When the image acquisition is complete, manually check that the autofocus of the cross-sections is correct and that the individual fibers are clearly distinguishable.It is critical that the autofocus is checked and corrected manually for every image as necessary. The automated muscle fiber quantification process requires high-quality images to obtain optimal results. Then, for every cross-section that should be included in the final analyses, export each channel except DAPI separately as JPEG files, and name and sort the files into the appropriately-named folders according to the exposed channels.Before beginning the muscle fiber quantification process, preview each image in the appropriate image-editing software to confirm a sufficient contrast between the stained fibers and the background. Next, in ImageJ, use the Plugins>Macros>Edit command to reveal the macro source code, and change the folder directory for each channel in the source code of the macro. Now, use the Run command to start the macro.All of the images in the folder will be loaded into the macro and quantified in a consecutive order. At the end of the quantification, export the results into the appropriate spreadsheet software, and identify the values for the positively-counted slow, intermediate, and fast fibers. The total fiber number can then be calculated.The immunofluorescent staining of muscle fibers is quick with minor cross-reactivity, and the resulting images demonstrate a high contrast between the stained fibers and the surrounding tissue, as well as a strong distinction between the different fiber types. Automatic analysis allows the detection of the relative muscle fiber populations with an accuracy of plus or minus 4%compared to the manual analysis, with the overall absolute counts higher compared to the manual analysis. The relative counts, however, remain similar, within a plus or minus 4%range.After watching this video, you should have a good understanding on how to use this method to automize muscle fiber quantification. Don't forget that working with DAPI can be hazardous. Therefore, wear the appropriate protective gear while performing the staining.

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11.2K Views.  Medical University of Vienna. The overall goal of this method is to rapidly and reliably guide muscle fiber quantification in whole rat muscles in an automatic and user-independent manner using an open-source platform. This method can help answer key questions in the neuromuscular field about defects of aging, disease, or trauma on skeletal muscles. The main advantage of this technique is that it can be used to reliably analyze muscle fiber populations and to generate high-quality images of muscle cross-sections.Af...