Name: Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique
Uploaded: 2023-09-22T14:15:00
Description: Watch this Scientific Journal Video about Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique at JoVE.com

The antibodies against MHC I (A4.840) and MHCIIa (A4.74) used in this study were developed by Dr. H. M. Blau and the antibody against MHCIIx (6H1) was developed by Dr. C. A. Lucas and obtained from the Developmental Studies Hybridoma Bank (DSHB), thanks to the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). We thank Victoria L. Wyckelsma for providing the human muscle samples for this study. The majority of the images in Figure 1 was sourced from BioRender.com.
Funding: 
This study received no external funding.

Human muscle samples were obtained from the vastus lateralis from n = 3 (2 males, 1 female), aged 70-74 years old under sterile conditions using local anesthesia (Xylocaine) and a Bergstrom needle modified for manual suction11,12. Samples were a subset of a previous study approved by the Victoria University Human Research Ethics Committee (HRETH11/221) and conducted in accordance with the Declaration of Helsinki13. Participants pr...

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

<ol>
	<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> Physiological Reviews. 91 (4), 1447-1531 (2011).</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), e35273 (2012).</li><li>Wyckelsma, V. L., 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=Cell+specific+differences+in+the+protein+abundances+of+GAPDH+and+Na(+),K(+)-ATPase+in+skeletal+muscle+from+aged+individuals.">Cell specific differences in the protein abundances of GAPDH and Na(+),K(+)-ATPase in skeletal muscle from aged individuals.</a> Experimental Gerontology. 75, 8-15 (2016).</li><li>Morales-Scholz, M. 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=Muscle+fiber+type-specific+autophagy+responses+following+an+overnight+fast+and+mixed+meal+ingestion+in+human+skeletal+muscle.">Muscle fiber type-specific autophagy responses following an overnight fast and mixed meal ingestion in human skeletal muscle.</a> American Journal of Physiology Endocrinology and Metabolism. 323 (3), e242-e253 (2022).</li><li>Tripp, T. 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=Time+course+and+fibre+type-dependent+nature+of+calcium-handling+protein+responses+to+sprint+interval+exercise+in+human+skeletal+muscle.">Time course and fibre type-dependent nature of calcium-handling protein responses to sprint interval exercise in human skeletal muscle.</a> The Journal of Physiology. 600 (12), 2897-2917 (2022).</li><li>Frankenberg, N. T., Mason, S. A., Wadley, G. D., Murphy, R. M. <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+cell-specific+differences+in+type+2+diabetes.">Skeletal muscle cell-specific differences in type 2 diabetes.</a> Cellular and Molecular Life Sciences. 79 (5), 256 (2022).</li><li>Murphy, R. M., 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=Activation+of+skeletal+muscle+calpain-3+by+eccentric+exercise+in+humans+does+not+result+in+its+translocation+to+the+nucleus+or+cytosol.">Activation of skeletal muscle calpain-3 by eccentric exercise in humans does not result in its translocation to the nucleus or cytosol.</a> Journal of Applied Physiology. 111 (5), 1448-1458 (2011).</li><li>Murphy, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+technique+to+measure+proteins+in+single+segments+of+human+skeletal+muscle+fibers:+fiber-type+dependence+of+AMPK-alpha1+and+-beta1.">Enhanced technique to measure proteins in single segments of human skeletal muscle fibers: fiber-type dependence of AMPK-alpha1 and -beta1.</a> Journal of Applied Physiology. 110 (3), 820-825 (2011).</li><li>Christiansen, 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=A+fast,+reliable+and+sample-sparing+method+to+identify+fibre+types+of+single+muscle+fibres.">A fast, reliable and sample-sparing method to identify fibre types of single muscle fibres.</a> Scientific Reports. 9 (1), 6473 (2019).</li><li>Skelly, L. E., 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=Human+skeletal+muscle+fiber+type-specific+responses+to+sprint+interval+and+moderate-intensity+continuous+exercise:+acute+and+training-induced+changes.">Human skeletal muscle fiber type-specific responses to sprint interval and moderate-intensity continuous exercise: acute and training-induced changes.</a> Journal of Applied Physiology. 130 (4), 1001-1014 (2021).</li><li>Bergstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+electrolytes+in+man.">Muscle electrolytes in man.</a> Scandinavian Journal of Clinical and Laboratory Investigation. (SUPPL. 68), 1-110 (1962).</li><li>Evans, W. J., Phinney, S. D., Young, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suction+applied+to+a+muscle+biopsy+maximizes+sample+size.">Suction applied to a muscle biopsy maximizes sample size.</a> Medicine &amp; Science in Sports &amp; Exercise. 14 (1), 101-102 (1982).</li><li>Wyckelsma, V. L., 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=Preservation+of+skeletal+muscle+mitochondrial+content+in+older+adults:+relationship+between+mitochondria,+fibre+type+and+high-intensity+exercise+training.">Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high-intensity exercise training.</a> The Journal of Physiology. 595 (11), 3345-3359 (2017).</li><li>Bortolotto, S. K., Stephenson, D. G., Stephenson, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+type+populations+and+Ca2+-activation+properties+of+single+fibers+in+soleus+muscles+from+SHR+and+WKY+rats.">Fiber type populations and Ca2+-activation properties of single fibers in soleus muscles from SHR and WKY rats.</a> American Journal of Physiology Cell Physiology. 276 (3), C628-C637 (1999).</li><li>Murphy, R. M., Lamb, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+considerations+for+protein+analyses+using+antibody+based+techniques:+down-sizing+Western+blotting+up-sizes+outcomes.">Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes.</a> The Journal of Physiology. 591 (23), 5823-5831 (2013).</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> Biochemical and Biophysical Research Communications. 272 (1), 303-308 (2000).</li><li>MacInnis, M. J., 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=Superior+mitochondrial+adaptations+in+human+skeletal+muscle+after+interval+compared+to+continuous+single-leg+cycling+matched+for+total+work.">Superior mitochondrial adaptations in human skeletal muscle after interval compared to continuous single-leg cycling matched for total work.</a> The Journal of Physiology. 595 (9), 2955-2968 (2017).</li></ol>

Fiber collection 
Based on several years of experience, most researchers can master this technique; however, practice leads to faster and more efficient fiber collection for downstream analyses. To be able to isolate 30 single fiber segments of a quality for pooling, it is recommended that 50 fiber segments are collected per sample. Studying the fiber collection video carefully and after performing two practice sessions (~50 fibers per session) is recommended to achieve a reasonable standard. All fi...

Skeletal muscle is a heterogeneous tissue, with distinct cellular metabolic and contractile properties that are dependent on whether the cell (fiber) is slow twitch (type I) or fast twitch (type II). The fiber type can be identified by examining the myosin heavy chain (MHC) isoforms, which differ from each other in several ways, including contraction time, speed of shortening, and fatigue resistance1. Major MHC isoforms include type I, type IIa, type IIb, and type IIx and their metabolic profiles are either oxidative (type I and IIa) or glycolytic (IIx, IIb)1. The proportion of these fiber types varies in muscle type and between species. Type IIb is widely found in rodent muscle. Human muscles do not contain any type IIb fibers and consist predominantly of MHC isoforms type I and IIa fibers, with a small proportion of IIx fibers2. Protein expression profiles vary between different fiber types and can be altered with aging3, exercise4,5, and disease6.
Measuring cellular responses in different skeletal muscle fiber types is often overlooked or not possible due to the examination of muscle homogenates (a mix of all fiber types). Single-fiber western blot allows for the investigation of multiple proteins in individual muscle fibers7. This methodology has previously been utilized to produce novel and informative single-fiber characteristics that were not possible to obtain using homogenate preparations. However, there are some limitations of the original single-fiber western blot methodology, including the time-consuming nature, the inability to generate sample replicates, and the use of expensive, sensitive enhanced chemiluminescence (ECL) reagents. If fresh tissue is being used, this method is further limited due to the time constraints of needing to isolate individual fibers within a limited timeframe (i.e., 1-2 h). Fortunately, this restraint is mitigated by isolating single-fiber segments from freeze-dried tissue8. However, fiber collection from freeze-dried samples is limited by the size and quality of the biopsied tissue.
Fiber type identification using the dot blotting method9 has been significantly elaborated upon and expanded in this comprehensive protocol. Previously, it has been demonstrated that as little as ~2-10 mg of wet weight muscle tissue is adequate for freeze-drying and single-fiber MHC isoform protein analysis9. Christensen et al.9 used 30% of a ~1 mm fiber segment to detect the MHC isoform present by dot blotting, which was confirmed by western blotting. This work showed that by substituting western blotting with dot blotting, the overall costs were reduced by ~40-fold (for 50 fiber segments). Fibers were then &#34;pooled&#34; into type I and type II samples, which allowed for experimental replication9. Nevertheless, a limitation was that only two fiber type-specific samples were obtained: type I (MHCI positive) and type II (MHCII positive fibers), with type II samples containing a mixture of MHCIIa and MHCIIx6,10. Notably, the current protocol demonstrates how pure type IIx fibers can be identified and provides a highly detailed workflow (summarized in Figure 1), including troubleshooting strategies for common protocol issues.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Denaturing buffer</td>
			<td>Make according to recipe</td>
			<td>Constituents can be sourced from Sigma-Aldrich or other chemical distributing companies</td>
			<td>&#160;1x denaturing buffer is made by diluting 3x denaturing buffer 1 in 3 v/v with 1X Tris-HCl (pH 6.8). Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>3x Denaturing buffer</td>
			<td>Make according to recipe</td>
			<td>Constituents can be sourced from Sigma-Aldrich or other chemical distributors</td>
			<td>3x denaturing buffer contains: 0.125M Tris-HCI, 10% glycerol, 4% SDS, 4 M urea, 10% 2-mercaptoethanol, and 0.001% bromophenol blue, pH 6.8.&#160; Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>N/A</td>
			<td>100% ethanol can be sourced from any company</td>
			<td>Diluted to 95% with ultra-pure H2O.</td>
		</tr>
		<tr>
			<td>Actin rabbit polyclonal antibody</td>
			<td>Sigma-Aldrich</td>
			<td>&#160; A2066</td>
			<td>Dilute 1 in 1,000 with BSA buffer.</td>
		</tr>
		<tr>
			<td>Analytical scales</td>
			<td>Mettler Toledo</td>
			<td>Model number: MSZ042101</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody enhancer</td>
			<td>Thermo Fischer Scientific</td>
			<td>32110</td>
			<td>Product name is Miser Antibody Extender Solution NC.</td>
		</tr>
		<tr>
			<td>Beaker (100 mL)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>5452</td>
			<td>Model name: Mini Spin.</td>
		</tr>
		<tr>
			<td>Blocking buffer: 5% Skim milk in Wash buffer.</td>
			<td>Diploma</td>
			<td>Store bought</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA buffer: 1 % BSA/PBST, 0.02 % NaN3</td>
			<td>BSA: Sigma-Aldrich&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; PBS: Bio-Rad Laboratories. NaN3 : Sigma-Aldrich&#160;</td>
			<td>BSA: A6003-25G&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 10x PBS: 1610780&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NaN3: S2002</td>
			<td>Bovine serum albumin (BSA), Phosphate-buffered saline (PBS), and Sodium azide (NaN3). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Cassette opening lever&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>4560000</td>
			<td>Used to open the precast gel cassettes.</td>
		</tr>
		<tr>
			<td>Chemidoc MP Imager&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>Model number: Universal hood III</td>
			<td>Any imaging system with Stain-Free gel imaging capabilities.</td>
		</tr>
		<tr>
			<td>Criterion blotter</td>
			<td>Bio-Rad Laboratories</td>
			<td>1704070</td>
			<td>Includes ice pack, transfer tray, roller, 2 cassette holders, filter paper, foam pads and lid with cables.</td>
		</tr>
		<tr>
			<td>Criterion Cell (Bio-Rad)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1656001</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL (enhanced chemiluminescence)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1705062</td>
			<td>Product name: Clarity Max Western ECL Substrate.</td>
		</tr>
		<tr>
			<td>Electrophoresis buffer 1x Tris Glycine SDS (TGS)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610772</td>
			<td>Dilute 10x TGS 1 in 10 with ultra-pure H2O.</td>
		</tr>
		<tr>
			<td>Filter paper, 0.34 mm thick</td>
			<td>Whatmann</td>
			<td>3030917</td>
			<td>Bulk size 3 MM, pack 100 sheets, 315 x 335 mm.</td>
		</tr>
		<tr>
			<td>Fine tissue dissecting forceps</td>
			<td>Dumont</td>
			<td>F6521-1EA</td>
			<td>Jeweller&#8217;s forceps no. 5.</td>
		</tr>
		<tr>
			<td>Flat plastic tray/lid&#160;&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Large enough to place the membrane on. Ensure the surface is completely flat.</td>
		</tr>
		<tr>
			<td>Freeze-drying System</td>
			<td>Labconco</td>
			<td>7750030</td>
			<td>Freezone 4.5 L with glass chamber sample stage.</td>
		</tr>
		<tr>
			<td>Freezer -80 oC&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any freezer with a constant temperature of -80 &#176;C is suitable.</td>
		</tr>
		<tr>
			<td>Gel releasers</td>
			<td>1653320</td>
			<td>Bio-Rad</td>
			<td>Slide under the membrane to gather or move the membrane.</td>
		</tr>
		<tr>
			<td>Grey lead pencil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Image lab software&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>N/A</td>
			<td>Figures refers to software version 5.2.1 but other versions can used.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Bio-Rad Laboratories</td>
			<td>1660521</td>
			<td>Any incubator that can be set to 37 &#176;C would suffice.</td>
		</tr>
		<tr>
			<td>Lamp</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer with flea</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane roller&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>1651279</td>
			<td>Can be purchased in the Transfer bundle pack. However, if this product is not available, any smooth surface cylindrical tube long enough to roll over the membrane would suffice.&#160;</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes&#160; (0.6 mL)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG HRP secondary</td>
			<td>Thermo Fisher Scientific</td>
			<td>31430</td>
			<td>Goat anti-Mouse IgG (H+L), RRID AB_228341. Dilute at 1 in 20,000 in blocking buffer.</td>
		</tr>
		<tr>
			<td>Mouse IgM HRP secondary</td>
			<td>Abcam</td>
			<td>ab97230</td>
			<td>Goat Anti-Mouse IgM mu chain. Use at the same dilution as mouse IgG.</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain I (MHCI) primary antibody</td>
			<td>DSHB</td>
			<td>A4.840&#160;</td>
			<td>Dilution range: 1 in 200 to 1 in 500 in BSA buffer.</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain IIa (MHCIIa) primary antibody</td>
			<td>DSHB</td>
			<td>A4.74&#160;</td>
			<td>Dilution range: 1 in 200 to 1 in 500 in BSA buffer.</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain IIx (MHCIIx) primary antibody</td>
			<td>DSHB</td>
			<td>6H1</td>
			<td>Dilution range: 1 in 200 to 1 in 500 in BSA buffer.</td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane 0.45 &#181;m&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>1620115</td>
			<td>For Western blotting.</td>
		</tr>
		<tr>
			<td>Petri dish lid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tweezers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Pack&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>164-5050</td>
			<td>Product name: Basic power supply.</td>
		</tr>
		<tr>
			<td>Protein ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>26616</td>
			<td>PageRuler Prestained Protein Ladder, 10 to 180 kDa.</td>
		</tr>
		<tr>
			<td>PVDF Membrane 0.2 &#181;m</td>
			<td>Bio-Rad Laboratories</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit HRP secondary</td>
			<td>Thermo Fisher Scientific</td>
			<td>31460</td>
			<td>Goat anti-Rabbit IgG (H+L), RRID AB_228341. Dilution same as mouse secondary antibodies.</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Motic</td>
			<td>SMZ-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Stripping buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>21059</td>
			<td>Product name: Restore Western Blot Stripping Buffer.</td>
		</tr>
		<tr>
			<td>Tissue (lint free)</td>
			<td>Kimberly-Clark professional</td>
			<td>34120</td>
			<td>Product name: Kimwipe.</td>
		</tr>
		<tr>
			<td>Transfer buffer (1x Tris Glycine buffer (TG), 20% Methanol)</td>
			<td>TG: Bio-Rad Laboratories&#160;&#160; Methanol: Merck</td>
			<td>TG buffer: 1610771&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Methanol: 1.06018</td>
			<td>dilute 10x TG buffer with ultra-pure H2O to 1x. Add 100% Methanol to a final concentration of 20% Methanol. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Transfer tray</td>
			<td>Bio-Rad Laboratories</td>
			<td>1704089</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;UV-activation precast gel</td>
			<td>Bio-Rad Laboratories</td>
			<td>5678085</td>
			<td>Gel type: 4&#8211;15% Criterion TGX Stain-Free Protein Gel, 26 well, 15 &#181;L.</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer (1x TBST)</td>
			<td>10x TBS: Astral Scientific&#160; Tween 20: Sigma</td>
			<td>&#160;BIOA0027-4L</td>
			<td>1x TBST recipe: 10x Tris-buffered saline (TBS) is diluted down to 1x with ultra-pure H2O, Tween 20 is added to a final concentration of 0.1%. Store buffer at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Wash containers</td>
			<td>Sistema</td>
			<td>Store bought</td>
			<td>Any tupperware container, that suits the approximate dimensions of the membrane would suffice.</td>
		</tr>
	</tbody>
</table>

fiber type identification of human skeletal muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

Fiber Type Identification of Human Skeletal Muscle

Our research centers around skeletal muscle, which is comprised of cells with distinctive metabolic and contractile properties, characterized as slow or fast twitch fibers. We delve into the factors contributing to these differences and explore their implications in the context of aging, exercise, and various diseases. Measuring cellular responses in different skeletal muscle fibers is often overlooked, or not possible due to the examination of muscle homogenates.The MyDoBID technique allows us to repeatedly analyze fiber type protein differences that were previously impossible to measure. This protocol accurately and efficiently identifies type one and type two muscle fibers from freeze dried human skeletal muscle samples. This newly revised method provides a straightforward approach for researchers to investigate protein differences in skeletal muscle at the cellular level.Notably, it facilitates the identification of type two X fibers, and these have historically been challenging to distinguish. The technique allows researchers to shed light on all fiber types, including the elusive type two X fiber. With the MyDoBID technique, numerous questions can now be answered.For example, one can discover the properties of type two X muscle fibers, and determine if there's evidence indicating preferential atrophy of type two X muscle fibers during aging or disease. One can also observe how a specific exercise intervention impacts each fiber type.

Identification of individual MHCI, MHCIIa, and MHCIIx muscle fibers using dot blotting 
A feature of MyDoBID is the categorization of the varying MHC and Actin signal intensity strength in a given fiber (Figure 4A). Fiber type was identified by the presence or absence of MHCI and IIa isoforms (Figure 4B). Six fibers showed no MHC or actin detection, indicating that there was no fiber collected. The results of this specific dot blot were the...

Watch this Scientific Journal Video about Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique at JoVE.com

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

fiber-type-identification-of-human-skeletal-muscle

Research

JoVE Journal

Biochemistry

Isolation of Single-Fiber from Freeze-Dried Human Skeletal Muscle

To begin, position the freeze dried muscle sample under the stereo microscope and view it at low magnification. With one pair of fine tissue dissecting forceps, hold the muscle in place and separate the small bundles of fibers with the other forceps. Isolate one bundle of fibers and gently tease them apart until single fiber segments are extracted from the bundle.Then shift the fiber to a clear area of the Petri dish. Investigate each single fiber segment under 50 times magnification. Attempt to further separate the fiber at one end.If the fiber begins to break, it is a single fiber. Carefully collect the fiber with a pair of forceps and place it into the previously prepared aliquot of denaturing buffer. Tap the tube firmly on the bench three times.Afterward, vortex the fiber samples and briefly centrifuge them for five seconds at 2, 500G to ensure the samples settles at the bottom of the tube. Let the samples rest at room temperature for one hour before storing them at minus 80 degrees Celsius for future use.

MHCI and MHCII Analysis of Single Fibers Isolated from Freeze-Dried Muscle Samples Using Dot Blot

To start, prepare the four sheets of filter paper, each measuring 12.5 by 7.5 centimeters. Stack two sheets together and place the stack in transfer buffer to soak through. Place the PVDF membrane in 95%ethanol and agitate the membrane on a rocker for one minute.Recollect the ethanol. Immerse the membrane in transfer buffer. And agitate for two minutes.Next, lay the soaking filter paper stack on a flat, movable surface, such as a large lid and flatten it using a roller. Using a gel releaser or tweezers, carefully position the PVDF membrane on the stack. Afterward, place a single sheet of dry filter paper on this membrane and glide the roller over the paper to absorb any excess buffer that's on the membrane's surface.Lift off the top filter paper without rubbing against the membrane. Take isolated fiber samples from the freezer and thaw them at room temperature before centrifuging and thoroughly re-suspending the sample. Place a one microliter droplet of each sample in the center of the designated membrane area.Avoid touching the membrane with the pipette tip. Allow the sample droplets to absorb entirely into the membrane for 15 minutes. Then using a gel releaser or tweezers, carefully lift the membrane from the damp filter paper stack.Place it onto a dry sheet of filter paper and leave it for at least five minutes to dehydrate. Reactivate the membrane after the sample spots have turned completely white. Then proceed with immunolabeling the target protein.Learn how to use the signal panel intensity to classify the intensity of the MHC isoforms and Actin signals from the dot blot images. Strong, medium and little target protein detection is shown by saturated, moderate and faint signals respectively. For fiber type identification, initially compare the myosin heavy chain IIA and myosin heavy chain one results.Document the fibers that display only a single myosin heavy chain isoform with either saturated or moderate signal intensity. Record the fibers detected with Actin and no detection of myosin heavy chain one or IIA as a potential type 2X. If a faint myosin heavy chain isoform signal is present with a moderate desaturated Actin signal, record it as unidentified.Discard samples with faint or undetected target proteins. Fibers displaying saturated or moderate signals of both MHCIIA and MHC1 should not be used for fiber type-specific preparation. After MYO1D ID, prepare type one and two samples by combining fibers with saturated or moderate signals for the respective MHC isoform.Use 10 microliters of each fiber type-specific sample and separate them on a precast gel by SDS page, following the manufacturer's guidelines. Use a gel imager to detect the target MHC isoform, following the manufacturer's protocol. Compare the signal intensity of each MHC isoform in all fiber type-specific samples.Fiber type ID is confirmed by the correct MHC isoform at moderate or saturated signal intensity. Immunolabeling of the dot blot allowed the identification of type two fibers, type one fibers and potential type 2X fibers. The fiber type-specific samples were validated, using western blotting and myosin heavy chain specific antibodies.