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>
	<li>Engel, W. 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. 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=Ca2++permeation+and/or+binding+to+Cav1.1+fine-tunes+skeletal+muscle+Ca2++signaling+to+sustain+muscle+function.">Ca2+ permeation and/or binding to Cav1.1 fine-tunes skeletal muscle Ca2+ signaling to sustain muscle function.</a> Skelet Muscle. 5 (4), (2015).</li><li>Sawano, 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=A+one-step+immunostaining+method+to+visualize+rodent+muscle+fiber+type+within+a+single+specimen.">A one-step immunostaining method to visualize rodent muscle fiber type within a single specimen.</a> PLoS One. 11 (11), (2016).</li><li>Meunier, B., Picard, B., Astruc, T., Labas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+image+analysis+tool+for+the+classification+of+muscle+fibre+type+using+immunohistochemical+staining.">Development of image analysis tool for the classification of muscle fibre type using immunohistochemical staining.</a> Histochem Cell Biol. 134 (3), 307-317 (2010).</li><li>Kammoun, M., Casser-Malek, I., Meunier, B., Picard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunohistochemical+classification+of+skeletal+muscle+fibres+in+mouse.">A simplified immunohistochemical classification of skeletal muscle fibres in mouse.</a> Eur J Histochem. 58 (2), 2254 (2014).</li><li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J Vis Exp. (99), (2015).</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 (197), (1989).</li><li>Augusto, V., Padovani, C. R., Campos, G. E. R. <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+fiber+types+in+C57Bl6j+mice.">Skeletal muscle fiber types in C57Bl6j mice.</a> Braz J Morphol Sci. 21 (2), 89-94 (2004).</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=Fiber+types+in+mammalian+skeletal+muscles.">Fiber types in mammalian skeletal muscles.</a> Physiol Rev. 91 (4), 1447-1531 (2011).</li><li>Allen, D. L., Harrison, B. C., Maass, A., Bell, M. L., Byrnes, W. C., Leinwand, L. 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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>
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			<td class="xl65" style="width:172pt" width="229">A9418-100G</td>
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			<td class="xl65" style="width:172pt" width="229">Discontinued</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>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<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="xl65" style="width:172pt" width="229"></td>
			<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" style="width:172pt" width="229">A21044</td>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">OCT</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 ...

The overall goal of this protocol is to use state-of-the-art immunohistochemistry to accurately and rapidly identify skeletal muscle fiber types. This method can help answer key questions in the field of muscle biology such as whether fiber type changes are precipitated by loss of functional proteins. Though this method can provide insight into many models of muscle disease, it can also be used in systems such as knock in and knock out models to reveal the greater impact of a single molecule on basic muscle function.To begin, air dry frozen sections of mouse muscle on charged slides for about half an hour. Once dried, draw a border around the sections on each slide using a hydrophobic barrier PAP pen. Next, to block nonspecific antibody binding, load about 250 microliters of 5%BSA in PBS within the drawn borders and incubate the slides at room temperature for an hour.Meanwhile, prepare a one to 50 dilution of the primary antibody in 5%BSA PBS. Make about 250 microliters per slide and store the antibody dilution on ice. After the block period, aspirate the block solution from the slides and add 250 microliter aliquots of the antibody dilution.For a negative control, apply 5%BSA in PBS without primary antibody. Next, load the Petri dish with dampened filter paper and cover it in aluminum foil for an incubation chamber. Then incubate the slides at four degrees Celsius for 24 to 48 hours in the chamber or in a similar environment with the same humidity.After the primary antibody incubation, aspirate the solution from the slides and wash the slides three times. For each wash, apply about 250 microliters of 5%BSA in PBS for about 10 minutes. While washing the slides, prepare one to 200 dilutions of the purified fluorophore conjugated secondary antibodies.Prepare at least 250 microliters per slide and keep the solution in the dark and on ice. After the third wash, add 250 microliters of the secondary antibody dilution to the slides. Then incubate the slides for 90 minutes at room temperature in a dark humid environment.After the incubation, aspirate the antibody solution and wash the slides three times as before. After washing away all of the excess secondary antibody, rinse the slides with PBS and allow them to air dry for 10 minutes. Lastly, mount the slides using a nonpermanent low viscosity aqueous mounting medium.After the medium dries, seal the edges of the cover slip with nail polish and transfer the slides into an opaque storage box. Before imaging a slide, clean the cover slip with 70%ethanol. Then obtain digital images using an epifluorescence microscope.Under low magnification, select a one square millimeter area of interest. To view Alexa 488 conjugated antibodies, use a 505 nanometer long pass filter. To view Alexa 594 conjugated antibodies, use a 595 nanometer long pass filter.With the filters properly set, start collecting digital images. Be sure to include a scale bar for subsequent analysis. For the analysis, install Fiji in the macro provided with this publication.Store the macro file in an easily accessed directory. Now, load a brightfield image of a selected section and navigate to the trainable weka segmentation option. In order to stipulate the cellular areas of the section, draw a line on a fiber and click add to class one.Then to mark the extracellular domains, draw a line on the space between fibers and click add to class two. Repeat this process until five to 10 labels of each class have been defined. It can be difficult to mark the interstitial boundaries.Using the zoom function allows one to mark these boundaries with greater precision. Next, select the train classifier option. On the resulting red and green overlay, manually add more labels to any parts of the image that were incorrectly segmented by the automated process.Do this until the fibers marked in red are appropriately separated from the intervening spaces marked in green. Then click on get probability and save the image in a new file folder. Now, repeat the entire process using the fluorescent image taken from the same field.Label the immunostained fibers as class one and all the nonfluorescent regions as class two. Ultimately, save the resulting probability map to the same folder where the processed brightfield image was stored. Now, open one of the previously saved probability maps in Fiji.Draw a straight line on the scale bar provided by the imaging software. Select set scale and input the correct values based on the scale bar. Then toggle the global option to standardize the scale to every image.Now, run the Tyagi macro for fiber quantification and the navigation pane will appear. Then open the images host folder. In the associated dialogue box, change the background dropdown value to light.The set measurements function can be used to define which parameters should be measured. CSA and feet diameter are the most pertinent measures to quantifying fiber morphology. After running the macro, the folder will then be populated with images of the fiber outlines and spreadsheets with the measurements made by the poor analysis function.Tibialis anterior and soleus muscles were analyzed using the described methods because they are mostly composed of either fast twitch or slow twitch fibers. Several different myosin heavy chains were identified using specific antibodies. A substantial fraction of the soleus muscle fibers expressed type one and type two A myosin heavy chains and a fair amount of the remaining fibers were positive for type two X.By contrast, virtually no type two B fibers were evident.By comparison, stained tibialis anterior muscles were composed mostly of type two B and type two X myosin heavy chain fibers with smaller contributions from fibers expressing type two A chains and virtually no fibers expressed in type one chains. Morphometric analysis of a tibialis anterior muscle probed with SC-71 antibodies directed to type two A myosin heavy chain provided several image transformations used to make probability maps. From this easy automated analysis, the data proved fully comparable to data generated using much more tedious manual calculations.All together, the analysis yield the data from about 6, 000 total individual fibers. The high data throughput made the statistical analysis far much more feasible. After watching this video, you should have a good understanding of how to immunostain muscle sections and use the provided semi-automated software to facilitate analysis of muscle fiber type and composition.While attempting this procedure, it is important to remember that muscle can be regionally heterogeneous and that analysis of many muscle fields is required for rigorous assessment of muscle fiber type and composition. In combination with this procedure, other methods like Trichome staining can be performed in order to answer additional questions like whether fibrosis develops in parallel with changes in fiber type composition.

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Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of ...