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.

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>

The authors have no conflicts of interest to declare.

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><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>&amp;nbsp;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 &amp;deg;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.&amp;nbsp; Store at -20 &amp;deg;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 H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Actin rabbit polyclonal antibody</td><td>Sigma-Aldrich</td><td>&amp;nbsp; 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 % NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>BSA: Sigma-Aldrich&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; PBS: Bio-Rad Laboratories. NaN&lt;sub&gt;3 &lt;/sub&gt;: Sigma-Aldrich&amp;nbsp;</td><td>BSA: A6003-25G&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10x PBS: 1610780&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; NaN&lt;sub&gt;3&lt;/sub&gt;: S2002</td><td>Bovine serum albumin (BSA), Phosphate-buffered saline (PBS), and Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;). Store at 4 &amp;deg;C.</td></tr><tr><td>Cassette opening lever&amp;nbsp;</td><td>Bio-Rad Laboratories</td><td>4560000</td><td>Used to open the precast gel cassettes.</td></tr><tr><td>Chemidoc MP Imager&amp;nbsp;</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 H&lt;sub&gt;2&lt;/sub&gt;O.</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&amp;rsquo;s forceps no. 5.</td></tr><tr><td>Flat plastic tray/lid&amp;nbsp;&amp;nbsp;</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 &lt;sup&gt;o&lt;/sup&gt;C&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Any freezer with a constant temperature of -80 &amp;deg;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>&amp;nbsp;Image lab software&amp;nbsp;</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 &amp;deg;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&amp;nbsp;</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.&amp;nbsp;</td></tr><tr><td>Microcentrifuge tubes&amp;nbsp; (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&amp;nbsp;</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&amp;nbsp;</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 &amp;micro;m&amp;nbsp;</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&amp;nbsp;</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 &amp;micro;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&amp;nbsp;&amp;nbsp; Methanol: Merck</td><td>TG buffer: 1610771&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Methanol: 1.06018</td><td>dilute 10x TG buffer with ultra-pure H&lt;sub&gt;2&lt;/sub&gt;O to 1x. Add 100% Methanol to a final concentration of 20% Methanol. Store at 4 &amp;deg;C.</td></tr><tr><td>Transfer tray</td><td>Bio-Rad Laboratories</td><td>1704089</td><td></td></tr><tr><td>&amp;nbsp;UV-activation precast gel</td><td>Bio-Rad Laboratories</td><td>5678085</td><td>Gel type: 4&amp;ndash;15% Criterion TGX Stain-Free Protein Gel, 26 well, 15 &amp;micro;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&amp;nbsp; Tween 20: Sigma</td><td>&amp;nbsp;BIOA0027-4L</td><td>1x TBST recipe: 10x Tris-buffered saline (TBS) is diluted down to 1x with ultra-pure H&lt;sub&gt;2&lt;/sub&gt;O, Tween 20 is added to a final concentration of 0.1%. Store buffer at 4 &amp;deg;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.

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

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

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.

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 ...

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

Research

JoVE Journal

Biochemistry

2.4K Views.  La Trobe University. 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.

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

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 ...

Medicine

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Biology

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

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 ...

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.

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 ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

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 ...

Transmission Electron Microscopy to Quantify Glycogen Distribution in Human Skeletal Muscles

Source: Jensen, R., et al. <a href="https://app.jove.com/v/63347">Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the sample preparation of human skeletal muscle tissue and its staining for transmission electron microscopy to visualize and quantify subcellular glycogen distribution.

Source: Jensen, R., et al. Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy. J. Vis. ...

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Classification of Skeletal Muscle Fibers

Skeletal muscles continuously produce ATP to provide the energy that enables muscle contractions. Skeletal muscle fibers can be categorized into three types based on differences in their contraction speed and how they produce ATP, as well as physical differences related to these factors. Most human muscles contain all three muscle fiber types, albeit in varying proportions.
<h4>Slow-Twitch Muscle Fibers</h4>
Slow oxidative, muscle fibers appear red due to large numbers of capillaries and high levels of myoglobin, an oxygen-storing protein. These fibers contain more mitochondria, which produce ATP through oxidative phosphorylation, than fast-twitch muscle fibers. 
Slow oxidative muscle fibers use aerobic respiration, involving oxygen and glucose, to produce ATP. In addition to contracting more slowly than fast-twitch fibers, the slow oxidative fibers receive nerve signals more slowly, contract for longer periods, and are more resistant to fatigue. These fibers primarily store energy as fatty substances called triglycerides.
<h4>Fast-Twitch Muscle Fibers</h4>
Relative to the slow oxidative fibers, the fast-twitch muscle fibers receive nerve signals and contract more quickly, but contract for shorter periods and fatigue more quickly. Fast-twitch muscle fibers primarily store energy as ATP and creatine phosphate.
Fast oxidative, muscle fibers primarily use aerobic respiration to produce ATP. However, they also use anaerobic respiration. Fast glycolytic, muscle fibers primarily use anaerobic respiration, which produces less ATP per cycle than aerobic respiration. Thus, Fast glycolytic fibers tire faster than the other fiber types.
More recently, scientists identified additional muscle fibers with characteristics intermediate between the above three types.

Skeletal muscles continuously produce ATP to provide the energy that enables muscle contractions. Skeletal muscle fibers can be categorized into three ...

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KEY TERMS AND CONCEPTS

Types of Skeletal Muscle Fibers

Skeletal muscles comprise various fibers, each with distinct characteristics and roles in movement and stability. They are mainly categorized into three types &#8212; fast-twitch, slow-twitch, and intermediate.
Fast-twitch fibers
Fast-twitch fibers, or Type II fibers, are designed for quick, powerful bursts of speed and strength. They reach peak tension within approximately 0.01 seconds following stimulation. Characterized by a large diameter and densely packed myofibrils, these fibers contain abundant glycogen reserves for rapid energy access. However, their relatively low mitochondrial content limits their endurance, making them prone to quick fatigue. Fast-twitch fibers primarily rely on anaerobic metabolism, consuming high levels of ATP quickly, and are best suited for short, intense activities like sprinting or heavy lifting.
Slow-twitch fibers
In contrast, slow-twitch fibers, or Type I fibers, are the endurance powerhouses of the muscle. With a smaller diameter, these fibers are packed with capillaries, high levels of myoglobin, and numerous mitochondria. This combination allows for efficient oxygen transport and utilization, supporting prolonged aerobic activity and ATP production. Slow-twitch fibers contract more slowly and can sustain contractions for extended periods, making them ideal for endurance activities like long-distance running or cycling. They are less reliant on anaerobic metabolism due to their enhanced oxygen reserves and blood supply.
Intermediate fibers
Intermediate fibers bridge the gap between fast and slow-twitch fibers. They resemble fast-twitch fibers in appearance, with minimal myoglobin content and a lighter color. However, they boast a more developed capillary network and a higher mitochondrial density than fast-twitch fibers. This endows them with greater resistance to fatigue and a capacity for both anaerobic and aerobic energy production. Intermediate fibers are adaptable and can change their characteristics with endurance or strength training, making them versatile components in various physical activities.

A skeletal muscle comprises different motor units, each containing slow or fast contracting fibers.
Slow fibers possess slow-functioning myosin ATPases, ...