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 et al.27 is available describing the correct freezing of muscle specimens in great detail.
<ol>
	<li>Obtain entire muscles or sufficient tissue to acquire entire cross sections immediately after euthanasia of the animals.
	<ol>
		<li>Remove the entire muscle from an anesthetized or euthanized rat. Remove all connective tissue and tendons surrounding the muscle with forceps and scissors. For anesthesia or euthanasia, follow the international guidelines of FELASA28.</li>
	</ol>
	</li>
	<li>Weigh muscle using a calibrated precision scale. This quick step allows comparative analyses between muscle samples, especially after interventional procedures.</li>
	<li>Put muscle into a vessel, filled entirely with O.C.T. compound, according to the size of the muscle with a safety margin of approximately 1 mm to all sides (e.g., made of aluminum foil).</li>
	<li>Freeze it in approximately 2 min precooled isopentane using liquid nitrogen. The isopentane must begin to show white crystals at the bottom of the container. 
	NOTE: This step is essential for preserving the correct architecture of the muscle and thereby allows the correct staining and fiber analyses27.</li>
	<li>Store samples conserved in O.C.T. for at least 24 h at -80 &#176;C. Samples can, however, be stored at -80 &#176;C for several months or even years, if correctly cooled.</li>
	<li>Cut 10 &#181;m transverse sections from the midportion of the muscle at -20 &#176;C using a cryotome. If cutting at 10 &#181;m is not feasible, increase the thickness in steps of 2 &#181;m up to a maximum of 20 &#181;m. Sufficiently sharp cutting blades are essential for this step.</li>
	<li>Apply the sections to adhesive slides by approximating the slides to the cross sections. The cross sections will automatically stick to the adhesive slides. Store the slides at -20 &#176;C overnight before staining.</li>
</ol>
2. Staining
NOTE: A large number of antibodies against myosin heavy chain proteins are available; however, high quality antibodies are essential for automated acquisition and analyses. For dilutions and reference to antibodies, see Table 1. For a spreadsheet file to calculate the correct dilutions and see the number of required solution quantities, see <a href="http://cloudfront.jove.com/files/ftp_upload/55441/Calculator_for_muscle_fiber_typing.xlsx">Supplemental File 1</a>.
<ol>
	<li>Thaw and air-dry the frozen muscle sections for 10 min before the staining procedure.</li>
	<li>Wash slides carefully with phosphate buffered saline (PBS) + 0.05% Triton X for 10 min and then for 5 min. Triton has been shown to improve the staining quality significantly. See the discussion for further details.</li>
	<li>Let sections air-dry for 2 min and outline individual cross sections on each slide using a hydrophobic pen (to minimize required amount of antibody) and allow additional drying for 15 min.</li>
	<li>Block all slides with PBS + 0.05% Triton X (PBST) containing 10% goat serum for 1 h at room temperature.</li>
	<li>Dilute all primary antibodies (Table 1) in a cocktail of PBS + 0.05% Triton X (PBST) containing 10% goat serum. Mix sufficiently before application. Triton X is essential for equal staining of the whole cross sections. Calculate approximately 30 &#181;L of primary antibody cocktail per cross section.</li>
	<li>For steps 2.7-2.13 ensure sufficient protection from light. Dimmed room lights and covered staining trays provide adequate protection. Additionally, fill the staining tray with fluid at the bottom to provide a humid environment during staining and prevent drying.</li>
	<li>Apply primary antibody (Table 1) cocktails to the slides for 1 h in a humid staining tray on room temperature.</li>
	<li>Wash slides with PBS + 0.05% Triton X for 10 min and then with new PBST for 5 min.</li>
	<li>Dilute secondary antibodies in a cocktail of PBS + 0.05% Triton X (PBST) + 10% goat serum. Calculate 30 &#181;L of primary antibody cocktail per cross section.</li>
	<li>Apply secondary antibody cocktail to the slides for 1 h.</li>
	<li>Wash slides with PBS + 0.05% Triton X (PBST) for 10 min and with new PBST for another 5 min.</li>
	<li>Apply a DAPI nuclear staining kit (preferably read-to-use solutions) for 15 min or according to the manufacturer&#39;s instructions to stain cell nuclei.</li>
	<li>Wash slides briefly with PBS + 0.05% Triton X (PBST) for 1 min. Afterward, let slides air-dry briefly.</li>
	<li>Apply fluorescent mounting medium and coverslips. Store at 4 &#176;C protected from light and perform imaging optimally within 24-48 h.</li>
</ol>
3. Microscopy
<ol>
	<li>Load slides into slide scanner. A maximum of 12 slides is feasible depending on the slide scanner.</li>
	<li>Start a new project. For preview set the DAPI channel and the 2.5X lens. For the detailed analysis use DAPI, GFP, FITC and Cy5 channels and the 20X objective. Autofocus is acquired throughout the project in the DAPI channel.</li>
	<li>Use the following colors for each channel: DAPI -blue, FITC: green, Texas Red: red, Cy5: yellow.</li>
	<li>Create previews of each slide using the DAPI channel. For this purpose, apply manual focus on the first specimen and use it throughout the preview analysis to acquire preview images of every specimen.</li>
	<li>Outline each cross-section that should be included for the detailed acquisition process. Do not draw outlines close to the edges of the specimen, to ensure caption of the entire area of interest.</li>
	<li>Set exposure times for each channel. In most cases, exposure parameters of the different channels are identical for all cross-sections.</li>
	<li>Run automatic image acquisition. Check correct acquisition for the first field of views, to prevent incorrect acquisition early in the automated process.</li>
	<li>If required, establish remote control using desktop mirroring software. This allows the remote monitoring of the image acquisition process by mirroring the computer screen of the slide scanner to a different computer via any network or internet connection. Although, no changes can be made to the physical setup regarding the used slides, the virtual environment of the image acquisition software allows monitoring the correct focus or exposure times of the images. In addition, the estimated time for completion can be checked regularly.</li>
	<li>After completion of the image acquisition, control the correct autofocus of all images by manually controlling if the fiber architecture, individual fibers and the various fiber types are distinguishable.</li>
	<li>Re-acquire incorrectly focused image areas for single fields of view or entire areas of interest. For reacquisition of single areas, mark areas or images throughout the entire project and reacquire areas with manual focus. In most cases, an additional image detail in a different focus level leads to the incorrectly focused images</li>
	<li>For every cross section that should be included in the final analyses, export each channel (excluding DAPI) separately as jpeg files. Name and sort files according to the exposed channels in folders named Texas Red, FITC and Cy5.</li>
</ol>
4. Automated Fiber Analysis
NOTE: The macro can be obtained from the following web page: https://www.meduniwien.ac.at/hp/bionicreconstruction/macro/
<ol>
	<li>Preview each image using a conventional image editing software before analyses and check for sufficient contrast between stained fibers and background. If necessary, adjust contrast and brightness to increase the difference, using brightness and contrast adjustment commands.</li>
	<li>Open ImageJ or Fiji. Open the macro using the command &#34;Plugins - Macros - Edit.&#34; This shows the macros source code and allows quick adaption of parameters. If necessary, change folder directory for each channel in the source code of the macro. 
	NOTE: Values for muscle fiber analyses are based on rat biceps and lumbrical muscles, other species or muscles may require different values.</li>
	<li>Use the &#34;run&#34; command to start the macro. All images of the folder are now loaded into the macro and quantified in a consecutive order. The results are shown in a new window. This step may take from seconds to several hours, depending on the amount of data being analyzed.</li>
	<li>Export the results window as a spreadsheet file. Identify values for positively counted Cy5 (slow), FITC (intermediate) and Texas Red (fast) fibers and sum up for total fiber number.</li>
</ol>

<ol>
	<li>Kung, T. A., 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=Motor+Unit+Changes+Seen+With+Skeletal+Muscle+Sarcopenia+in+Oldest+Old+Rats.">Motor Unit Changes Seen With Skeletal Muscle Sarcopenia in Oldest Old Rats.</a> J Gerontol A Biol Sci Med Sci. 69 (6), 657-665 (2014).</li><li>Greising, S. M., Medina, J. S., Vasdev, A. K., Sieck, G. C., Mantilla, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+muscle+fiber+clustering+in+the+diaphragm+muscle+of+sarcopenic+mice.">Analysis of muscle fiber clustering in the diaphragm muscle of sarcopenic mice.</a> Muscle Nerve. 52 (1), 76-82 (2015).</li><li>Claflin, D. 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=Effects+of+high-+and+low-velocity+resistance+training+on+the+contractile+properties+of+skeletal+muscle+fibers+from+young+and+older+humans.">Effects of high- and low-velocity resistance training on the contractile properties of skeletal muscle fibers from young and older humans.</a> J Appl Physiol. 111 (4), 1021-1030 (2011).</li><li>Miller, A. I., Heath, E. M., Dickinson, J. M., Bressel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+Between+Muscle+Fiber+Type+and+Reactive+Balance:+A+Preliminary+Study.">Relationship Between Muscle Fiber Type and Reactive Balance: A Preliminary Study.</a> J Mot Behav. 47 (6), 497-502 (2015).</li><li>Song, Y., Forsgren, S., Liu, J. -. X., Yu, J. -. G., St&#229;l, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+Muscle+Overuse+Causes+Bilateral+Changes+in+Muscle+Fiber+Composition+and+Vascular+Supply.">Unilateral Muscle Overuse Causes Bilateral Changes in Muscle Fiber Composition and Vascular Supply.</a> PLoS ONE. 9 (12), 116455 (2014).</li><li>Hopker, J. 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=The+influence+of+training+status,+age,+and+muscle+fiber+type+on+cycling+efficiency+and+endurance+performance.">The influence of training status, age, and muscle fiber type on cycling efficiency and endurance performance.</a> J Appl Physiol (1985). 115 (5), 723-729 (2013).</li><li>Pette, D., Staron, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+isoforms,+muscle+fiber+types,+and+transitions.">Myosin isoforms, muscle fiber types, and transitions.</a> Microsc Res Tech. 50 (6), 500-509 (2000).</li><li>Suga, 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=Muscle+fiber+type-predominant+promoter+activity+in+lentiviral-mediated+transgenic+mouse.">Muscle fiber type-predominant promoter activity in lentiviral-mediated transgenic mouse.</a> PLoS One. 6 (3), 16908 (2011).</li><li>Wang, J. F., Forst, J., Schroder, S., Schroder, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+muscle+fiber+type+measurements+with+clinical+and+molecular+genetic+data+in+Duchenne+muscular+dystrophy.">Correlation of muscle fiber type measurements with clinical and molecular genetic data in Duchenne muscular dystrophy.</a> Neuromuscul Disord. 9 (3), 150-158 (1999).</li><li>Rader, E. P., 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=Effect+of+cleft+palate+repair+on+the+susceptibility+to+contraction-induced+injury+of+single+permeabilized+muscle+fibers+from+congenitally-clefted+goat+palates.">Effect of cleft palate repair on the susceptibility to contraction-induced injury of single permeabilized muscle fibers from congenitally-clefted goat palates.</a> Cleft Palate Craniofac J. 45 (2), 113-120 (2008).</li><li>Macaluso, F., Isaacs, A. W., Myburgh, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+type+II+muscle+fiber+damage+from+plyometric+exercise.">Preferential type II muscle fiber damage from plyometric exercise.</a> J Athl Train. 47 (4), 414-420 (2012).</li><li>Lieber, R. L., Frid&#233;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+of+skeletal+muscle+architecture.">Clinical significance of skeletal muscle architecture.</a> Clin. Orthop. Relat. Res. 383, 140-151 (2001).</li><li>Schiaffino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibre+types+in+skeletal+muscle:+a+personal+account.">Fibre types in skeletal muscle: a personal account.</a> Acta Physiol (Oxf). 199 (4), 451-463 (2010).</li><li>Bottinelli, R., Betto, R., Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unloaded+shortening+velocity+and+myosin+heavy+chain+and+alkali+light+chain+isoform+composition+in+rat+skeletal+muscle+fibres.">Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres.</a> J Physiol. 478, 341-349 (1994).</li><li>Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+isoforms+in+mammalian+skeletal+muscle.">Myosin isoforms in mammalian skeletal muscle.</a> J Appl Physiol (1985). 77 (2), 493-501 (1994).</li><li>Larsson, L., Moss, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+velocity+of+shortening+in+relation+to+myosin+isoform+composition+in+single+fibres+from+human+skeletal+muscles.">Maximum velocity of shortening in relation to myosin isoform composition in single fibres from human skeletal muscles.</a> J Physiol. 472, 595-614 (1993).</li><li>Kostrominova, T. Y., Reiner, D. S., Haas, R. H., Ingermanson, R., McDonough, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+methods+for+the+analysis+of+skeletal+muscle+fiber+size+and+metabolic+type.">Automated methods for the analysis of skeletal muscle fiber size and metabolic type.</a> Int Rev Cell Mol Biol. 306, 275-332 (2013).</li><li>Schiaffino, 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=Three+myosin+heavy+chain+isoforms+in+type+2+skeletal+muscle+fibres.">Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.</a> J Muscle Res Cell Motil. 10 (3), 197-205 (1989).</li><li>Lieber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Skeletal muscle structure, function, and plasticity. , (2009).</li><li>Hintz, C. S., Coyle, E. F., Kaiser, K. K., Chi, M. M., Lowry, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+muscle+fiber+typing+by+quantitative+enzyme+assays+and+by+myosin+ATPase+staining.">Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining.</a> J Histochem Cytochem. 32 (6), 655-660 (1984).</li><li>Havenith, M. G., Visser, R., van Schendel, J. M. S. c. h. r. i. j. v. e. r. s. -., Bosman, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+fiber+typing+in+routinely+processed+skeletal+muscle+with+monoclonal+antibodies.">Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies.</a> Histochemistry. 93 (5), 497-499 (1990).</li><li>Likar, B., Pernu&#353;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registration+of+serial+transverse+sections+of+muscle+fibers.">Registration of serial transverse sections of muscle fibers.</a> Cytometry. 37 (2), 93-106 (1999).</li><li>Liu, F., 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=Automated+fiber-type-specific+cross-sectional+area+assessment+and+myonuclei+counting+in+skeletal+muscle.">Automated fiber-type-specific cross-sectional area assessment and myonuclei counting in skeletal muscle.</a> J Appl Physiol (1985). 115 (11), 1714-1724 (2013).</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), 35273 (2012).</li><li>Bergmeister, K. D., 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=Automated+muscle+fiber+type+population+analysis+with+ImageJ+of+whole+rat+muscles+using+rapid+myosin+heavy+chain+immunohistochemistry.">Automated muscle fiber type population analysis with ImageJ of whole rat muscles using rapid myosin heavy chain immunohistochemistry.</a> Muscle Nerve. 54 (2), 292-299 (2016).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+guidelines+and+recommendations.">FELASA guidelines and recommendations.</a> J Am Assoc Lab Anim Sci. 51 (3), 311-321 (2012).</li><li>Meng, H., 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=Tissue+Triage+and+Freezing+for+Models+of+Skeletal+Muscle+Disease.">Tissue Triage and Freezing for Models of Skeletal Muscle Disease.</a> J Vis Exp. (89), e51586 (2014).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+Guidelines+and+Recommendations.">FELASA Guidelines and Recommendations.</a> J Am Assoc Lab Animal Sci. 51 (3), 311-321 (2012).</li><li>Ribari&#269;, S., &#268;eba&#352;ek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Visualization+of+Myosin+Heavy+Chain+Isoforms+in+Single+Muscle+Sections.">Simultaneous Visualization of Myosin Heavy Chain Isoforms in Single Muscle Sections.</a> Cells Tissues Organs. 197 (4), 312-321 (2013).</li></ol>

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><tbody><tr><td>O.C.T compound</td><td>Tissue-Tek, Sakura, Netherlands</td><td></td><td>For embedding of muscle tissue</td></tr><tr><td>Isopentane</td><td></td><td></td><td>for adequate freezing of muscle tissue</td></tr><tr><td>Superfrost Ultra Plus slides</td><td>Thermo Scientific, Germany</td><td>1014356190</td><td>adhesive slides</td></tr><tr><td>phosphate buffered saline&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific, Germany</td><td>85112</td><td>Detergent Soluation</td></tr><tr><td>Goat serum</td><td>Thermo Scientific, Germany</td><td>50197Z</td><td>Goat Serum</td></tr><tr><td>DAKO Fluorescent Mounting Medium</td><td>Dako Denmark</td><td>S3023</td><td></td></tr><tr><td>Dako pen</td><td>Dako Denmark</td><td>S200230-2</td><td></td></tr><tr><td>TissueFAXSi plus</td><td>TissueGnostics, Vienna, Austria</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>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><td>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><td>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><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa&amp;nbsp;Fluor&amp;nbsp;633&amp;nbsp;Goat Anti-Mouse IgG2b&amp;nbsp;</td><td>Thermo Scientific, Germany</td><td>A-21146</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG1&amp;nbsp;(&amp;gamma;1)</td><td>Thermo Scientific, Germany</td><td>A-21121</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse IgM (&amp;micro; chain)</td><td>Thermo Scientific, Germany</td><td>A-21426</td><td></td></tr><tr><td>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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JoVE Journal

Biology

11.3K Views.  Medical University of Vienna.  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. 

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

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Medicine

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

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

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

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

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Neuroscience

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Confocal Microscopic Analysis of Rat Muscle Mitochondria

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

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions.
Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT).&#160;The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

Author Spotlight: Mitochondrial Remodeling in Skeletal Muscle 

Here, we present a protocol to analyze changes in mitochondrial density and longitudinal distribution by live-skeletal muscle imaging using confocal microscopy for mitochondrial network scanning.

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

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

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

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