Name: semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition
Uploaded: 2017-08-31T13:00:00
Description: Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

We are grateful to the Boettcher Foundation and the Amyotrophic Lateral Sclerosis Association (#17-II-344) for their support of this research.

All procedures involving mice were approved by the University of Colorado-Anschutz Medical Campus Institutional Animal Care and Use Committee (91813(05)1D).
1. Day 1: Primary (1&#176;) Immunostaining with Bovine Serum Albumin (BSA) Blocking
<ol>
	<li>Air-dry frozen sections of mouse hindlimb muscle (e.g., tibialis anterior, soleus) mounted on charged slides for ~ 30 min11. Draw a border around the sections using a hydrophobic barrier PAP pen.</li>
	<li>Place ...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essentiality+of+histo-+and+cytochemical+studies+of+skeletal+muscle+in+the+investigation+of+neuromuscular+disease.">The essentiality of histo- and cytochemical studies of skeletal muscle in the investigation of neuromuscular disease.</a> Neurol. 12, 778-784 (1962).</li><li>Dobrowolny, 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=Skeletal+muscle+is+a+primary+target+of+SOD1G93A-mediated+toxicity.">Skeletal muscle is a primary target of SOD1G93A-mediated toxicity.</a> Cell Metab. 8, 425-436 (2008).</li><li>Sultana, N., 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=Restricting+calcium+currents+is+required+for+correct+fiber+type+specification+in+skeletal+muscle.">Restricting calcium currents is required for correct fiber type specification in skeletal muscle.</a> Development. 143 (9), 1547-1559 (2016).</li><li>Guth, L., Samaha, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+the+histochemical+demonstration+of+actomyosin+ATPase.">Procedure for the histochemical demonstration of actomyosin ATPase.</a> Exp Neurol. 28 (2), 365-367 (1970).</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), (2012).</li><li>Lucas, C. A., Kang, L. H., Hoh, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monospecific+antibodies+against+the+three+mammalian+fast+limb+myosin+heavy+chains.">Monospecific antibodies against the three mammalian fast limb myosin heavy chains.</a> Biochem Biophys Res Commun. 272 (1), 303-308 (2000).</li><li>Lee, C. 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Here, we have provided useful direction for the identification of skeletal muscle fiber types. In doing so, we describe a novel algorithm for analysis of the data.
Since our results largely confirm those of previous reports5,8,10 and reflect our own manual measurements, the algorithm appears to be accurate. Still, we encountered some infrequent experimental pitfalls including unclear fiber borders res...

It has been known for some time that skeletal muscle is composed of single fibers of many types1. Initially, two groups of fibers were characterized based on their contractile properties and named, appropriately, slow-twitch (type I) and fast-twitch (type II). These categories were further distinguished on the basis of fiber metabolism. Since type I fibers are rich in mitochondria and reliant on oxidative metabolism, they were elucidated by robustly positive nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) diaphorase2 or succinate dehydrogenase (SDH)3 staining. By contrast, type II fibers exhibited lesser and variable degrees of NADH-TR diaphorase or SDH staining and were divided into two fast-twitch subgroups (type IIA and type IIB) somewhat crudely based on their relative oxidative capacities. These distinctions between fibers have been visualized more effectively by myosin-ATPase staining where type I fibers stain dark after a pre-incubation at pH 4.0 and type IIB fibers absorb precipitate following pre-incubation at pH 10.0 with type IIA fibers staining intermediately4.
More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type5. Type I, type IIA and type IIB fibers can be all identified with precision based on their MyHC profile. In addition, another fast-twitch metabolically-intermediate fiber type, type IIX, has been identified6. Hybrid fibers expressing more than one MyHC have also been confirmed5,7,8. Some species such as the cat and the baboon are known not to express Type IIB MyHCs6. Though MyHC immunostaining is currently the state-of-the-art assessment of muscle composition, the analysis of the data obtained via this technique is cumbersome and time-consuming without automated assistance. To this end, a handful of semi-automated methods to analyze these data have been developed5,9,10. Here, we present a relatively standard protocol for immunohistochemical identification of muscle fiber-type5,7,8,10, along with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology with accuracy.

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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Bovine Serum Albumin</td>
			<td class="xl65" style="width:140pt" width="187">Sigma Aldrich</td>
			<td class="xl65" style="width:172pt" width="229">A9418-100G</td>
			<td class="xl66" style="width:193pt" width="257">5% in PBS</td>
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			<td class="xl65" height="41" style="width:226pt;height:30.75pt" width="301">Hydrophobic Barrier Pap Pen</td>
			<td class="xl65" style="width:140pt" width="187">Scientific Device Laboratory</td>
			<td class="xl65" style="width:172pt" width="229">9804-02</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Microscope Slides</td>
			<td class="xl65" style="width:140pt" width="187">Globe Scientific</td>
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			<td class="xl65" height="41" style="width:226pt;height:30.75pt" width="301">Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope</td>
			<td class="xl65" style="width:140pt" width="187"></td>
			<td class="xl65" style="width:172pt" width="229">Discontinued</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Dell Optiplex</td>
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			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
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			<td class="xl70">monoclonal mouse IgG2b; 1:50</td>
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			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">SC-71 Primary Antibody</td>
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			<td class="xl70">monclonal mouse IgG1; 1:50</td>
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			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
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			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">6H1 Primary Antibody</td>
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			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgG2b</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgGM</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21044</td>
			<td class="xl66">goat anti-mouse;1:200</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">OCT</td>
			<td class="xl65" style="width:140pt" width="187">Sakura Finetek</td>
			<td class="xl68" style="width:172pt" width="229">4583</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">isopentane</td>
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			<td class="xl65" style="width:140pt" width="187">Fisher Bioreagents</td>
			<td class="xl65" style="width:172pt" width="229">BP665-1</td>
			<td class="xl66">10X, dilute to 1X</td>
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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.

Hindlimb muscles (i.e., tibialis anterior, soleus) dissected from a male C57BL/6 mouse of unknown age were flash frozen by sinking a plastic mold containing the muscle in OCT compound in liquid nitrogen-cooled isopentane. Then, using a cryotome, 8-10 &#181;m serial sections were cut at -20 &#176;C and transferred to different positively-charged glass slides12.
We chose tibialis anterior and soleu...

Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

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

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 ...

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