We thank Michaela Rau, Lea-Fedia Rissmann, Michael Marsh, Janina-Sophie Tennler, Kilian Kimmeskamp, and Wolfgang Linke for assisting with the project. Funding for this project was provided by the MERCUR Foundation (ID: An-2016-0050) to DH.

NOTE: Below, we outline a protocol to harvest mechanically undamaged fibers from the TA of volunteers who were enrolled in a separate ongoing study. This protocol is similar to that described by Shanely et al.3, who have described the modified Bergstr&#246;m technique in vastus lateralis. The information presented here has been refined by our research group but may not be ideal for all lab groups or organizational setups. We give only guidelines, and strongly suggests that laboratories new to biopsy collection consult experienced laboratory groups before attempting any human trials.
All studies conducted in this paper were approved by the Ethics Committee of the Faculty of Sport Science at Ruhr University Bochum. Participants gave free written informed consent prior to participating in the study.
1. Experimental preparation
<ol>
	<li>Assess exclusion criteria while taking the participant&#8217;s detailed medical history during the participant consultation (see below).
	<ol>
		<li>Exclude participants if they suffered an injury to the target muscle during the 6 weeks leading up to the biopsy. Ensure participants are generally healthy, aware of no muscle or coagulation disorders, and are not currently on medication that causes blood thinning (e.g., aspirin). 
		NOTE: Here, we selected participants who were moderately active and instructed them to refrain from intensive or unaccustomed leg exercises at least 3 days before the biopsy. However, for other research questions, these criteria may change.</li>
	</ol>
	</li>
	<li>Adhere to sterilization and aseptic techniques, as regulated by German law and common practice and overseen by the team physician22,23. This procedure can often be conducted as a &#8220;bedside&#8221; procedure or in an outpatient surgical suite. Consult the local regulatory body for guidance.</li>
	<li>Compose the biopsy team. We suggest that the biopsy team includes 4 people. A physician (or trained individual in biopsy collection), one medical assistant working with the physician, one assistant who monitors and interacts with the participant, and one assistant who handles the muscle biopsy immediately after extraction. With these numbers, quick patient care can be administered if a medical emergency occurs during the procedure. If comfortable with the procedure, then the team could be made of only two people: the physician and medical assistant, who would together take on patient care and tissue processing concurrently.</li>
	<li>Have the participant meet with the project lead/physician to review, discuss, and sign the user consent form. Take a detailed medical history (allergies, injuries or surgeries to the lower limb and TA) and exclude the participant if they meet any of the exclusion criteria. Thoroughly discuss recovery and incision hygiene.
	<ol>
		<li>Explain to the participant that they will be sore but able to walk around immediately after the procedure; walking down slopes or stairs is often uncomfortable for the first 48 hours, with full activity usually returning after 72 hours. Finally, explain that, to limit infection and mechanical abrasions, the incision site should remain bandaged for at least 1 week and kept clean.</li>
	</ol>
	</li>
</ol>
2. Visualize the Anterior Tibialis with B-mode Ultrasound
<ol>
	<li>Instruct the participant to lie down in a comfortable supine position and relax their leg muscles as much as possible. Use a custom-made device (see below) or have the assistant hold the ankle in a slightly dorsiflexed position to mimic that which will be done during the biopsy. 
	NOTE: It is important that the participant has a relaxed TA so that it replicates the muscle characteristics during the procedure. During the exam, ask the participant to contract and relax the muscle so that the changes in muscle architecture can be noted.</li>
	<li>Use an ultrasound probe to visualize the superficial and deep compartments of the TA, to survey the muscle architecture and decide on depth of insertion and needle angle of attack (Figure 2A-B). Indicate landmarks on the skin.
	<ol>
		<li>Give particular attention to the selection of a target area that avoids major veins, arteries, or nerves.</li>
		<li>Assess the cross section of the muscle, with the goal of identifying the central aponeurosis within TA muscle belly (approximately 1/3 of the leg, distal to the knee, and 2 cm lateral of the tibial crest) (Figure 2B). Record the location and depth of the central aponeurosis (usually 1.5-3 cm) so that care can be taken to not drive the collection (Bergstr&#246;m) needle past this point.</li>
		<li>Position the ultrasound probe in the proximal-distal orientation over the target location and visualize the fascicle pennation and muscle thickness (Figure 2A). Use this information to help successfully drive (blindly) the collection needle into the muscle belly. Save images of the target site in both planes for future reference during the surgical procedure.</li>
	</ol>
	</li>
	<li>With this information, create a plan for needle movement towards the target area.
	<ol>
		<li>Plan to make the incision 1-3 cm distal from the target biopsy area. After the needle is passed into the muscle, rotate the needle to a ~45% angle to the skin along the long axis of the limb, and then driven proximally towards the biopsy area. This strategy limits the chance of driving the needle into the central aponeurosis, if the needle is pushed too hard. Furthermore, the needle can be driven distally or proximally, depending on the handedness of&#160;the needle operator.</li>
	</ol>
	</li>
</ol>
3. Biopsy procedure
<ol>
	<li>Instruct the participant to lay supine on the operating table and relax their leg muscles. Make sure the participant&#8217;s line of sight to the biopsy site is blocked by a curtain.
	<ol>
		<li>Remove passive tension from the muscle belly by placing the participant&#8217;s limb into a device that fixes the ankle into a slightly dorsiflexed position (0-5&#176; from neutral; Figure 3). Ask the patient if they can still relax their muscle, as too much dorsiflexion can potentially make it difficult to relax. 
		NOTE: We have found that collecting biopsies from a dorsiflexed foot, no more than 5&#176; of neutral (i.e., the sole of the foot perpendicular to the shank) produces more consistent and larger biopsies than more plantar flexed ankle angles. The device that keeps the ankle dorsiflexed is a custom-made device. However, any number of (cheap) devices can be fabricated that still produce the desired result.</li>
	</ol>
	</li>
	<li>Shave, clean, and disinfect the selected incision area, as per standard practices24. 
	NOTE: The participant&#8217;s &#8220;clean&#8221; area is about 20 cm proximal-distal and 10 cm medial-lateral of the proposed incision site. However, always consult the institution&#8217;s and/or national regulations (if any) on this topic. The disinfection protocol includes scrubbing the skin clean and then disinfecting four times with liberal use of medical-grade disinfection spray. If the participant leaves the table for any reason, the disinfection protocol must be restarted.</li>
	<li>Administer a suprafascial injection of 1.5 cc of 2% Xylocitin with Epinephrine at the biopsy site, which functions as a local anesthetic and vasoconstrictor. Wait for the allotted affect time of ~20-30 min. 
	NOTE: These drugs are myotoxic and thus must never be injected into the muscle, only the subcutaneous tissue. As a reaction to the vasoconstriction, the area of the injection site may turn white (on lighter skin tones) or gray (darker skin tones).</li>
	<li>Confirm drug effect with skin pitches and gentle pokes with a sterile scalpel.</li>
	<li>At the previously marked biopsy site, make a 1 cm proximal-distal incision with a sterile scalpel that cuts through the skin and fascia, exposing the muscle belly. Take care to cut the fascia fully because the needle is blunt and will not pass through the fascia.</li>
	<li>Push the biopsy needle 0.5-1.0 cm into the muscle with an orientation perpendicular to the skin (Figure 2C, 2E). 
	NOTE: The operator will feel a change in the tension needed to drive the needle through the different tissue types. The fatty tissue is easy, the fascia is the toughest, and the muscle is in between (but can be variable, based on the participant).</li>
	<li>Orient the needle to a position of ~45&#176; angle to the skin, along the long axis of the leg (Figure 2D, 2F). Push the needle another 1-2 cm into the muscle until the needle tip is at the target location within the muscle. 
	NOTE: The physician should utilize the saved ultrasound images to account for individual variation of muscle dimensions. Because the incision is only large enough to insert the needle, the physician drives the needle blindly through the skin. There is a &#8220;feel&#8221; that the biopsy operator gains with experience. A novice must learn the skill from a trained biopsy operator (more on this in the discussion).</li>
	<li>Attach the 100 mL syringe and hose to the biopsy needle (Figure 1G). Apply suction to the Bergstr&#246;m needle by pulling the plunger of the syringe by about 15-20 mL to produce a negative pressure in the needle and sucking the muscle tissue into the needle window. Then, excise the muscle by a quick push(es) of the trocar over the needle window. 
	NOTE: Before and during suction, it is sometimes helpful to place light pressure on the skin immediately above the needle window to help push the muscle into the needle.</li>
	<li>Gently remove the needle from the leg, rotating slowly. There should only be light resistance while extracting the needle. If there is more resistance, this may indicate a partial biopsy cut. It this occurs, return the need to the target location, and reattempt tissue collection.</li>
	<li>Push the excised tissue towards the needle window using the internal ramrod.</li>
	<li>Carefully remove the sample from the needle. 
	NOTE: Submerging the needle into collection solution (see fiber preparation section) often dislodges the biopsy from the needle. Additionally, the syringe can be used to drive air through the needle and push out the sample. These techniques remove the need to physically touch the biopsy with tweezers and reduces the possibility of damage. If tools, hands (gloved or not) or non-sterile solutions come in contact with the needle, the needle cannot be used furthering during the procedure. Thus, if a second immediate biopsy is needed, then a new sterile needle must be used. This often occurs, so it is a best practice to maintain several sterile needles in reserve.</li>
	<li>Identify the tissue as muscle and not adipose or connective tissue. Muscle tissue is easily identified from other tissue because of its deep red color (Figure 4A). Sometimes, the collected tissue is not muscle, but fat or connective tissue.
	<ol>
		<li>If an adequate amount of muscle tissue is collected, continue the protocol. If there is not enough muscle, attempt the biopsy again.</li>
		<li>If a second biopsy is needed, carefully monitor the participant, as a second needle push occasionally makes the participant more uncomfortable than the first.</li>
	</ol>
	</li>
	<li>Wash muscle samples immediately in a collection solution and prepare for single fiber experiments (see muscle biopsy handling and storage).
	<ol>
		<li>Have an experienced assistant check the sample quality (see below) and assess the need to perform a second biopsy. A separate assistant takes the biopsy for processing, while the rest of the team continues with the participant.</li>
	</ol>
	</li>
	<li>Close the incision site.
	<ol>
		<li>Close the incision wound with sterile Leukostrip tape. Use one or more pieces to join the edges of the incision site by laying them perpendicular to the long axis of the incision, and then lay further strips in a starshaped pattern to protect against multi-directional loading. 
		NOTE: Proper handling of this step will reduce scarring. Suturing the wound can be done but is not necessary. Other options include wound glue.</li>
		<li>Place sterile wound dressing (e.g., Leucomed T plus) over the incision site to protect against infection.</li>
		<li>Wrap the leg with cohesive elastic bandages (e.g., Unihaft) to limit initial bleeding and protect against external mechanical impact.</li>
		<li>Wrap the leg with acrylastic compression bandages to prevent bleeding and protect the deeper bandages from becoming loose or destroyed.</li>
	</ol>
	</li>
</ol>
4. Post-biopsy care
<ol>
	<li>Ask the participant to walk around immediately after the procedure. There will be localized soreness. Instruct the participant to walk as normally as possible.</li>
	<li>Instruct the participant to not remove the bandages or let water soak the bandages. They must be kept on for at least: one days for the acrylastic bandage, three days for the cohesive elastic bandage, and seven days for the wound dressing. Inform the participant that they can be rebandaged if needed.
	<ol>
		<li>Tailor the post-biopsy care of a participant to the needs of the individual. Have a trained assistant or physician assess the participant and make an appropriate post-biopsy care plan. For this procedure, we suggest that any further in vivo neuromuscular testing of the TA is separated by at least a week from the biopsy.</li>
	</ol>
	</li>
</ol>
5. Muscle biopsy handling and storage
<ol>
	<li>After tissue extraction, immediately place the tissue into a 5 mL vial containing rigor collection solution (in mM: Tris (50), KCl (2), NaCl (100), MgCl2 (2), EGTA (1), protease inhibitor tablet (1), pH 7.0) and lightly shake for 4-6 min to wash out blood.</li>
	<li>Exchange the Rigor solution for fresh rigor, lightly shake for 4-6 min, and then store at 4 &#176;C for 4-6 h to allow the exchange of protease-inhibitor storage solution and blood.</li>
	<li>Exchange Rigor solution for overnight rigor (in mM: Tris (50), KCl (2), NaCl (100), MgCl2 (2), EGTA (1), protease inhibitor tablet (1), 50:50 glycerol, pH 7.0), and store at 4 &#176;C for 12-18 h.</li>
	<li>Exchange overnight rigor for 50:50 collection rigor:glycerol and stored at -20 &#176;C for up to 3 months, or one year in a -80 &#176;C freezer. 
	NOTE: This process permeabilizes the fiber membrane which allows for manual addition of calcium into and out of the cell. This process takes time and could be different between different muscles and species.</li>
</ol>

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A., 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=Suction-modified+needle+biopsy+technique+for+the+human+soleus+muscle.">Suction-modified needle biopsy technique for the human soleus muscle.</a> Aviation, Space, and Environmental Medicine. 84 (10), 1066-1073 (2013).</li><li>Edwards, R. H., Round, J. M., Jones, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Needle+biopsy+of+skeletal+muscle:+a+review+of+10+years+experience.">Needle biopsy of skeletal muscle: a review of 10 years experience.</a> Muscle &amp; Nerve. 6 (9), 676-683 (1983).</li><li>Gibreel, W. O., 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=Safety+and+yield+of+muscle+biopsy+in+pediatric+patients+in+the+modern+era.">Safety and yield of muscle biopsy in pediatric patients in the modern era.</a> Journal of Pediatric Surgery. 49 (9), 1429-1432 (2014).</li><li>Cuisset, J. 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=Muscle+biopsy+in+children:+Usefulness+in+2012.">Muscle biopsy in children: Usefulness in 2012.</a> Revue Neurologique. 169 (8-9), 632-639 (2013).</li><li>Nilipor, Y., 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=Evaluation+of+one+hundred+pediatric+muscle+biopsies+during+a+2-year+period+in+mofid+children+and+toos+hospitals.">Evaluation of one hundred pediatric muscle biopsies during a 2-year period in mofid children and toos hospitals.</a> Iranian Journal of Child Neurology. 7 (2), 17-21 (2013).</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> Physiological Reviews. 91 (4), 1447-1531 (2011).</li><li>Wang, K., Wright, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+sarcomere+matrix+of+skeletal+muscle:+immunoelectron+microscopic+evidence+that+suggests+a+set+of+parallel+inextensible+nebulin+filaments+anchored+at+the+Z+line.">Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line.</a> The Journal of Cell Biology. 107 (6), 2199-2212 (1988).</li><li>Ma, W., Gong, H., Irving, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+head+configurations+in+resting+and+contracting+murine+skeletal+muscle.">Myosin head configurations in resting and contracting murine skeletal muscle.</a> International Journal of Molecular Sciences. 19 (9), (2018).</li><li>Ma, W., Gong, H., Kiss, B., Lee, E. J., Granzier, H., Irving, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thick-Filament+Extensibility+in+Intact+Skeletal+Muscle.">Thick-Filament Extensibility in Intact Skeletal Muscle.</a> Biophysical Journal. 115 (8), 1580-1588 (2018).</li><li>Bonafiglia, J. T., 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+comparison+of+pain+responses,+hemodynamic+reactivity+and+fibre+type+composition+between+Bergstr&#246;m+and+microbiopsy+skeletal+muscle+biopsies.">A comparison of pain responses, hemodynamic reactivity and fibre type composition between Bergstr&#246;m and microbiopsy skeletal muscle biopsies.</a> Current Research in Physiology. 3, 1-10 (2020).</li><li>Wickiewicz, T. L., Roy, R. R., Powell, P. L., Edgerton, 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=Muscle+architecture+of+the+human+lower+limb.">Muscle architecture of the human lower limb.</a> Clinical Orthopaedics and Related Research. (179), 275-283 (1983).</li></ol>

The authors have nothing to disclose.

In this report, we described a technique for the biopsy of structurally undamaged muscle tissue from TA. We found that this procedure yields an acceptable content of usable muscle fibers (5-10 fiber bundle preparations per 50 mg of collected tissue) for mechanical testing. Further, we had enough tissue for follow-up mechanical, genetic, and proteomic experiments.
There are several methods typically used for the collection of muscle biopsies3,<sup class="xref"...

The study of human muscle physiology for clinical or research purposes often requires muscle biopsies. For example, a major challenge in human muscle physiology and biomechanics is to distinguish between and understand the various adaptations of muscle performance to exercise. Performance adaptations do not just include structural adaptations (e.g., changes in contractile proteins, muscle architecture) but also include neural adaptations1, which are very hard, if not impossible, to assess separately when testing intact in situ human muscles. Fiber-level experiments remove these higher-order components and allow for a more direct evaluation of muscle contraction and can be collected via biopsy techniques. Muscle biopsies have been collected since at least 18682. Today, the predominant technique to collect muscle biopsies is the modified Bergstr&#246;m technique3,4,5, although other techniques are available including the use of a Weil-Blakesley conchotome6 or the so called fine-needle7,8. All these techniques use special needle-like instruments that are designed to pass into muscle and cut a piece of tissue. Specifically, the modified Bergstr&#246;m technique uses a large modified needle (5 mm needle size here; Figure 1) that has a window close to the needle tip and a smaller internal trocar that moves up and down the needle, cutting the muscle when passing over the needle window. Within this hallow trocar is a ramrod that moves up and down the shaft of the trocar and pushes the biopsy towards the needle window. To pull the muscle into the needle window, a suction hose is attached, which sucks air out of the needle and pulls the muscle into the needle window via negative pressure.
Muscle biopsies are often acquired to study changes in protein content, gene expression, or morphology caused by disease or in a response to an exercise program1,9,10,11. Another critical use for muscle biopsies is mechanical experiments such as the measurement of fiber contractile force, muscle fiber stiffness, and history-dependent muscle properties12,13,14,15,16. Single fiber or fiber bundle mechanics are measured by attaching fibers between a length motor and force transducer on specialized rigs that control fiber length while simultaneously measuring force. By permeabilizing (e.g., skinning) fibers, the sarcolemma membrane becomes permeable to chemicals in the bath solution, allowing for activation control by varying calcium concentration. Furthermore, the effect of contractile properties on chemicals/pharmaceuticals/other proteins can easily be evaluated by adding the reagent in question to the bath solution. However, while this technique is highly used in other animal models, noticeably fewer studies conducted mechanical tests on skinned fibers from human muscle biopsies17,18,19. One reason is that the biopsy tools and protocols are designed to remove as much muscle tissue as possible with less regard for the level of structural damage sustained during tissue extraction. Indeed, a recent biopsy protocol suggests to drive the biopsy needle into the muscle and collect 2-4 chunks of muscle3. The process itself does little damage to the DNA or protein material, but often destroys fiber and sarcomeric structures in such a way that the activation of muscle fibers becomes unstable or impossible. Furthermore, the relative length of fibers within the biopsy are typically short (&#60;2 mm) and not easily handled for mechanical testing. For mechanical testing, ideal fibers are long (3-5 mm) and not structurally damaged.
More advanced tissue extraction techniques can be used to limit fiber damage. For example, one group20 took advantage of previously planned &#8220;open surgeries&#8221; of forearms (e.g., bone fracture repair), where the muscles were fully exposed and a surgeon was able to visualize the muscle structure and carefully dissect relatively large and structurally undamaged samples of muscle tissue (15 mm x 5mm x 5 mm). This &#8220;open biopsy&#8221; technique is favored when participants are undergoing a previously planned procedure, and so limits the pool of potential participants, especially for healthy adults, where no surgeries would otherwise take place. Thus, many biopsies conducted for research purposes are done as an outpatient procedure and the incision site is kept as small as possible to limit infection risk, scarring, and healing time. Therefore, most biopsies are collected blindly (i.e., the operator is unable to see the collection needle as it passes through the fascia into the muscle). This implies that the quality of the biopsy is almost entirely based on the skill and experience of the operator. Every muscle has its own difficulties when collecting tissue, such as risks to violate nerves and blood vessels, selection of an ideal collection depth and location, and deciding on an appropriate body position to keep the muscle as slack as possible. Unfortunately, most of the muscle-specific skillsets are not written down and so each physician must &#8220;reinvent the wheel&#8221; when performing biopsies on muscles new to them. This lack of experience usually leads to several collections with low quality until the physician identifies the best practices for biopsies on that muscle. Novice physicians often learn the skill through conversations with their more experienced colleagues, but relatively few informative and peer-reviewed texts exist on the matter, especially for muscles that are not traditionally used for biopsy collection. If we consider the above information, along with the difficulty of recruiting human volunteers for biopsies, it is clear that more teaching information is needed that maximizes the chances of success for every participant.
Thus, the purpose of this paper was to present a muscle biopsy technique that provides protocols for the successful collection of muscle biopsies with long, undamaged fiber fragments for mechanical tests. Human muscle biopsies are usually carried out on, and the bulk of biopsy training material is on, the musculus vastus lateralis. Its relatively large muscle size and superficial location relative to the skin allows for the collection of adequate muscle tissue, while minimizing patient discomfort and physical trauma1,21. However, there are some limitations to using the vastus lateralis for longitudinal training studies. For example, during experimental protocols that include a training program, participants must refrain from additional training outside of the study for a period that often spans 2-6 months. For athletes, this is often not possible, as the vastus lateralis is usually trained during typical exercises (e.g., squats, jumps), or is generally used for the sport (e.g., running, cycling). These separate training experiences away from the study&#8217;s aim can cause muscular adaptations that alter muscle mechanics, architecture, and physiology in such a way that it is difficult or impossible to know the true effect of the study&#8217;s experimental protocol on muscle properties. For these types of studies, it would be ideal to select a target muscle that is often not the focus of training regiments. The musculus tibialis anterior (TA) is an ideal target muscle that satisfies the requirements above. In addition, training interventions can be targeted towards the TA using controllable approaches, such as with the use of a dynamometer. There is almost no training material pertaining to a TA muscle biopsy. Therefore, we developed a modified protocol to collect relatively undamaged muscle biopsies from the TA.

<table>
	<tbody>
		<tr>
			<td>26 guage subcutaneous needle with 2 ml glass syringe</td>
			<td>B. Braun Melsungen AG 
			Carl-Braun-Stra&#223;e 1 
			34212 Melsungen, Hessen 
			Germany 
			&#160;</td>
			<td>4606027V</td>
			<td>Drug administration</td>
		</tr>
		<tr>
			<td>5mm Berst&#246;m needle</td>
			<td>homemade</td>
			<td>N/A</td>
			<td>Tissue collection. Similar to other Berst&#246;m needles</td>
		</tr>
		<tr>
			<td>Acrylastic</td>
			<td>BSN medical GmbH 
			22771 Hamburg</td>
			<td>269700</td>
			<td>elastic compression bandage</td>
		</tr>
		<tr>
			<td>Complete protease inhibitor cocktail</td>
			<td>Roche Diagnostics, Mannheim, Germany</td>
			<td>11836145001</td>
			<td>Protease inhibitor tabeletes added to all solutions that hold muscle tissue.</td>
		</tr>
		<tr>
			<td>Cutasept</td>
			<td>PAUL HARTMANN AG 
			Paul-Hartmann-Stra&#223;e 12 
			89522 Heidenheim 
			Germany</td>
			<td>9805630</td>
			<td>Disenfectant spray for the skin</td>
		</tr>
		<tr>
			<td>Leucomed T plus</td>
			<td>BSN medical GmbH 
			22771 Hamburg</td>
			<td>7238201</td>
			<td>Transparent wound dressing with wound pad to seal the wound and protect against infection</td>
		</tr>
		<tr>
			<td>Leukostrip</td>
			<td>Smith and Nephew medical Limitied 101 Hessle road, 
			Hull 
			Great Britain</td>
			<td>66002876</td>
			<td>wound closure</td>
		</tr>
		<tr>
			<td>Surgical disposable scalpels</td>
			<td>Aesculap AG 
			Am Aesculap-Platz 
			78532 Tuttlingen 
			Germany</td>
			<td>BA200 series</td>
			<td>Incision</td>
		</tr>
		<tr>
			<td>Unihaft cohesive elastic bandage</td>
			<td>BSN medical GmbH 
			22771 Hamburg</td>
			<td>4589600</td>
			<td>cohesive elastic bandage that protects against mechanical impact</td>
		</tr>
		<tr>
			<td>Xylocitin 2% with Epinephrin</td>
			<td>Milbe GmbH 
			M&#252;nchner Stra&#223;e 15 
			06796 Brehna 
			Germany</td>
			<td>N/A</td>
			<td>Controlled substance anesthesia, vasoconstriction</td>
		</tr>
	</tbody>
</table>

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.

The entire time commitment for a participant was about one hour (10 min consultation, 10 min ultrasound, 20 min surgery preparation and anesthetic administration, 10 min surgery, and 10 min recovery). Often, participants unconsciously activated their TA and needed consistent reminders to keep the muscle as relaxed as possible. When the biopsy needle was inside the muscle, participants usually reported a unique &#8220;pressure&#8221; sensation in the area around the biopsy needle, with occasional periods of moderate to in...

Watch this Scientific Journal Video about Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation at JoVE.com

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

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 ...

collection-skeletal-muscle-biopsies-from-superior-compartment-human

Research

JoVE Journal

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6.7K Views.  Ruhr University Bochum. This technical report describes a variation of the modified Bergström technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

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

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

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 ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.