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

Vi er taknemmelige for instituttets Boettcher og foreningen Amyotrofisk Lateral sklerose (#17-II-344) for deres støtte til denne forskning.

alle procedurer, der involverer mus blev godkendt ved University of Colorado-Anschutz Campus institutionelle dyr lægebehandling og brug Udvalget (91813(05)1D).
 1. dag 1: primære (1°) immunfarvning med bovint serumalbumin (BSA) blokering <ol><li>lufttørre frosne dele af mus hindlimb muskel (fx, tibialis anterior, soleus) monteret på ladet dias for ~ 30 min 11. tegne en kant omkring sektioner ved hjælp af en hydrofobe barriere PAP pen.</li>
	<li>Sted ~...

<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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Her har vi givet nyttige retning til identifikation af skelet muskel fibertyper. Dermed, beskriver vi en roman algoritme til analyse af data.
Da vores resultater i vid udstrækning bekræfte dem i tidligere rapporter5,8,10 og afspejler vores egen manuelle målinger, synes algoritmen, der at være præcis. Stadig, vi stødte på nogle sjældne eksperimentelle faldgruber herunder uklart fiber grænser s...

Det har været kendt i nogen tid, at skeletmuskulaturen består af enkelt fibre af mange typer1. I første omgang to grupper af fibre blev karakteriseret baseret på deres kontraktile egenskaber og ved navn, passende, langsom-spjæt (type I-) og fast-spjæt (type II). Disse kategorier blev yderligere skelnes på grundlag af fiber stofskifte. Type I-siden fibre er rige på mitokondrier og afhængige af oxidative metabolisme, de blev belyst ved håndfast positive nikotinamidadenindinucleotid tetrazolium reduktase (NADH-TR) diaphorase2 eller succinat dehydrogenase (SDH)3 farvning. Derimod type II fibre udstillet mindre og varierende grader af NADH-TR diaphorase eller SDH farvning og var opdelt i to fast-spjæt undergrupper (type IIA og type IIB) lidt groft baseret på deres relative oxidative kapacitet. Disse sondringer mellem fibre har været visualiseret mere effektivt af myosin ATPase pletter når type I fibre pletten mørke efter en præ-inkubation ved pH 4,0 og type IIB fibre absorbere bundfald følgende præ-inkubation ved pH 10,0 med type IIA fibre farvning intermediately4.
Mere nylig, immunhistokemisk farvning for myosin heavy chain (MyHC) isoformer fremstod som en finere diskriminator af fiber-type5. Type I, type IIA og type IIB fibre kan alle identificeres med præcision baseret på deres MyHC profil. Desuden en anden fast-spjæt metabolisk mellemliggende fiber type, type IIX, er blevet identificeret6. Hybrid fibre giver udtryk for mere end én MyHC er også blevet bekræftet5,7,8. Nogle arter som katten og bavian er kendt for ikke at udtrykke Type IIB MyHCs6. Selvom MyHC immunfarvning er i øjeblikket den state-of-the-art vurdering af muskel sammensætning, er analyse af data hentet via denne teknik besværlige og tidskrævende uden automatiseret hjælp. Med henblik herpå, en håndfuld af semi-automatiserede metoder til at analysere disse data har været udviklede5,9,10. Vi præsenterer her, et relativt standard-protokol til immunhistokemiske identifikation af muskel fiber-type5,7,8,10, sammen med en semi-automatiske roman algoritme, der accelererer analyse af fiber-type og fiber morfologi med nøjagtighed.

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semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition

I år, var sondringer mellem skelet muskel fibertyper bedst visualiseret ved myosin ATPase pletter. Mere nylig, immunhistokemisk farvning for myosin heavy chain (MyHC) isoformer fremstod som en finere diskriminator af fiber-type. Type I, type II, type IIX og type IIB fibre kan nu blive identificeret med præcision baseret på deres MyHC profil; men manuel analyse af disse data kan være langsom og ned-højre kedelig. I denne henseende, er hurtig, nøjagtig vurdering af fiber-type sammensætning og morfologi en meget ønskelig værktøj. Vi præsenterer her, en protokol til state-of-the-art immunhistokemisk farvning af MyHCs i frosne dele fremstillet af mus hindlimb muskel i koncert med en roman semi-automatiske algoritme, der accelererer analyse af fiber-type og fiber morfologi. Som forventet, den soleus muskel vises farvning type I og type IIA fibre, men ikke for type IIX eller type IIB fibre. På den anden side tibialis anterior musklen bestod overvejende af type IIX og type IIB fibre, en lille brøkdel af type II fibre og ringe eller ingen type I-fibre. Flere billede transformationer blev brugt til at generere sandsynlighed kort med henblik på måling af forskellige aspekter af fiber morfologi (dvs.tværsnitsareal (CSA), maksimal og minimal Feret diameter). De værdier, der er opnået for disse parametre blev derefter sammenlignet med manuelt opnåede værdier. Ingen væsentlige forskelle blev observeret mellem enten tilstand af analyse med hensyn til CSA, maksimal eller minimal Feret diameter (alle p &#62; 0,05), der angiver nøjagtigheden af vores metode. Således, vores immunfarvning analyse protokollen kan anvendes til undersøgelse af effekter på muskel sammensætning i mange modeller af aldring og myopathy.

Hindlimb muskler (dvs., tibialis anterior, soleus) dissekeret fra en mandlig C57BL/6 mus af ukendt alder var flash frosset ved at sænke en plast støber indeholdende musklen i OLT sammensatte i flydende nitrogen-kølet isopentane. Derefter, ved hjælp af en cryotome, 8-10 µm seriel afsnit skåret på-20 ° C og overført til forskellige positivt ladede glas dias12.
Vi valgte tibialis anterior o...

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

Immunhistokemisk farvning for myosin heavy chain isoformer fremstod som den state-of-the-art diskriminator skelet muskel fiber-type (dvs., type I-, type II, type IIX, type IIB). Her præsenterer vi en farvning protokol sammen med en roman semi-automatiske algoritme, der letter hurtig vurdering af fiber-type og fiber morfologi.

Semi-automatiseret analyse af musen skeletmuskulatur morfologi og Fiber-type sammensætning

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

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