We thank the NIAMS Light Imaging Section for providing the microscopes and technical help. The MF20 and Pax7 antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences, The University of Iowa, Iowa City. This work was supported by the Intramural Research Program of NIAMS of the National Institutes of Health.

In this protocol, the anterior hind limb muscles of adult mice (2-6 months), tibialis anterior (TA) and extensor digitorum longus (EDL), were employed as an example to perform the immunofluorescence staining on their SCs. All the steps handling mice and muscle tissue dissections have been approved by the Animal Care and Use Committee (ACUC) of NIAMS/NIH.
1. Dissect the TA/EDL Muscle from the Mouse Hind Limb
<ol>
	<li>Euthanize the mouse in a CO2 filled euthanasia chamber under the guidelines of the ACUC of the NIH. Perform cervical dislocation if needed.</li>
	<li>Put the mouse face-up on a dissection pad (supine). Spray 70% ethanol onto its belly skin. Cut a 1-2 cm opening horizontally on the center of the mouse belly skin, then deskin the mouse by pulling the opening toward the mouse feet. Expose one side of the mouse hind limb.</li>
	<li>Use two sharp forceps: use one to hold the tendons that attach the TA/EDL to the ankle, and use the other one to break and shear the fascia covering the TA/EDL. Once the fascia is peeled off, use the sharp tip of the forceps to detach the TA/EDL from the tibia bone by running the tip underneath the TA/EDL muscle.</li>
	<li>Cut off the ankle tendons with scissors while still holding them with the forceps. Trim the muscle with scissors along the longitudinal line of the TA/EDL from the ankle to the knee while lifting the TA/EDL from the ankle side with the forceps. Cut the knee-side tendons to detach the whole TA/EDL from the tibia.</li>
</ol>
2. Cryo-embedding of the TA/EDL Muscle
<ol>
	<li>Prepare a liquid nitrogen bath in a container, e.g., a liquid nitrogen Dewar flask. Pour methylbutane into a small plastic beaker (about 20 mL) to half-full. Then put the beaker in the liquid nitrogen bath to freeze it. After a few minutes, check that the methylbutane is frozen. 
	NOTE: The liquid nitrogen can be replaced by a bucket of dry ice, but the freezing time is slower.</li>
	<li>Lay the dissected TA/EDL onto the flat surface of a small plastic syringe plunger. Fully cover the TA/EDL with a few drops of Optimal Cutting Temperature (O.C.T.) compound (the freezing medium).</li>
	<li>Take the methylbutane beaker out of the liquid nitrogen bath and thaw the surface with a warm object (e.g., the handle of scissors). Quickly dip the O.C.T. covered TA/EDL on the plunger into the melted methylbutane to snap-freeze the tissue.</li>
	<li>Detach the embedded tissue from the plunger with forceps, and put it into a 1.5 mL microcentrifuge tube. Place the tube into liquid nitrogen to store it while handling more samples. 
	NOTE: The embedded tissue can be stored at -80 &#176;C for long term (2 years) or -20 &#176;C for short term (6 months).</li>
</ol>
3. Cryostat Sectioning
<ol>
	<li>Add a drop of O.C.T. compound onto the center of a specimen holder of a cryostat. Wait until the O.C.T. compound is almost solidified. Quickly stick the embedded TA/EDL to the O.C.T. compound, perpendicularly with the ankle-side down and the knee-side up.</li>
	<li>Mount the specimen holder onto a cryostat, cut the tissue at 10 &#181;m interval, and collect the cryo-sections on frost coated slides with about 4-8 sections on one slide.</li>
	<li>Dry the sections at room temperature for at least 3 h to overnight. 
	NOTE: Although drying the sections in the freezer for overnight or longer is acceptable, it will inevitably increase the autofluorescence. For best results, dry the sections for 0.5 h, then proceed with the fixation and staining steps immediately.</li>
	<li>Collect the slides in a slide box and use them immediately, or store the slides at -80 &#176;C or -20 &#176;C for later use.</li>
</ol>
4. Preparation of Reagents for Immunofluorescence Staining
<ol>
	<li>Prepare buffers:
	<ol>
		<li>Prepare 2 L of 1x phosphate buffered saline (PBS) from 10x PBS.</li>
		<li>Prepare 40 mL of 4% paraformaldehyde (PFA) in 1x PBS from 16% PFA.</li>
		<li>Prepare 2 L of PBST: 1x PBS with 0.1% Triton-100.</li>
		<li>Prepare 50 mL of PBST-B: PBST with 2% bovine serum albumin (BSA).</li>
		<li>Prepare 10 mL of PBST-B-G: PBST with 2% BSA and 5% Normal Goat Serum.</li>
		<li>Prepare 1 mL of Blocking solution. Dilute AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L) (1:10) in PBST-B.</li>
		<li>Prepare 200 mL of 1x citrate buffer. 
		NOTE: The volumes of solutions above are sufficient for at least 5 slides. Volumes should be adjusted according to the number of slides and the size of the slide containers.</li>
	</ol>
	</li>
	<li>Prepare antibody stains:
	<ol>
		<li>Primary Antibodies (working concentration): Prepare 1.2 mL of primary antibody mix by diluting Pax7 monoclonal mouse antibody (IgG1) (1-5 &#181;g/mL) + Laminin polyclonal rabbit antibody (0.5-2 &#181;g/mL) + MF20 monoclonal mouse antibody (IgG2b) (0.5-2.5 &#181;g/mL) in PBST-B-G. 
		NOTE: MF20 monoclonal mouse antibody (IgG2b) is optional.</li>
		<li>Secondary Antibodies (working concentration): Prepare 1.2 mL of secondary antibody mix by diluting Goat anti-Mouse IgG1 cross-adsorbed secondary antibody, Alexa Fluor 488 (0.5-2 &#181;g/mL) + Goat anti-Rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (0.5-2 &#181;g/mL) + Goat anti-Mouse IgG2b cross-adsorbed secondary antibody, Alexa Fluor 647 (0.5-2 &#181;g/mL) in PBST-B-G. 
		NOTE: Alexa Fluor 647 is optional.</li>
	</ol>
	</li>
</ol>
5. Immunofluorescence Staining Steps
<ol>
	<li>Rehydrate and fix the cryo-sections with PFA.
	<ol>
		<li>Remove a few of the TA/EDL cryo-section slides from the slide box. Warm the slides in a slide-holding tray at room temperature.</li>
		<li>Use a liquid blocker pen (PAP pen) to draw an area that includes all the cryo-sections on the slide. This circle will prevent the solutions from flowing off the slide.</li>
		<li>Take the tray to a laboratory fume hood. Add 300-500 &#181;L of 4% PFA onto the slide within the circled area to fix the sections at room temperature for 10 min. Wash out the PFA with 1x PBS at least 3x 5 min in a slide container. 
		CAUTION: PFA is toxic and a hazardous waste; it should be operated with care and disposed into a hazardous waste container.</li>
	</ol>
	</li>
	<li>Antigen retrieval
	<ol>
		<li>Fill another slide container with 200 mL of citrate buffer.</li>
		<li>Transfer the slides into the container. Transfer the container with the slides to a pressure cooker to cook the slides in high pressure mode for 10 min.</li>
		<li>Cool down the container with draining water for 10 min. Transfer all slides to a new container with PBST (200 mL), rinse the slides with PBST for at least 3x 5 min.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Add water-soaked kimwipes to the tray holding the slides to create a moist environment to avoid drying of the slides.</li>
		<li>Move the slides back to the tray, and add 200 &#181;L of blocking solution onto each slide. Cover the tray with a cap and let the blocking develop for 30 min. 
		NOTE: Pax7 antibody is a mouse monoclonal antibody, so there is unspecific binding of mouse IgG in mouse muscle tissues that creates high background. Using the AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L) blocks the mouse-on-mouse unspecific binding.</li>
	</ol>
	</li>
	<li>Apply primary antibodies.
	<ol>
		<li>Rinse the slides with PBST once in the slide container. Then move the slides back to the staining tray.</li>
		<li>Apply 200 &#181;L of primary antibody mix on each slide, cover the tray with a cap, and let the reaction develop at room temperature for 1 h or at 4 &#176;C for overnight. 
		NOTE: The overnight reaction at 4 &#176;C gives the best result, although developing the reaction at room temperature gives satisfactory results. The myosin MF20 antibody can be added in the mix to perform a triple (Pax7, Laminin, MF20) labeling. MF20 stains differentiated muscle fibers. This step also serves as another blocking step with PBST-B-G to reduce potential unspecific binding from secondary antibodies (goat antibodies) in the following step.</li>
	</ol>
	</li>
	<li>Apply secondary antibodies.
	<ol>
		<li>Wash out the primary antibodies with PBST at room temperature at least 3x 5 min.</li>
		<li>Add 200 &#181;L of secondary antibody mix to each slide, and allow the reaction to develop at room temperature for at least 1 h. 
		NOTE: For Pax7+Laminin, use Goat Anti-Mouse IgG1 Alexa 488 and Goat Anti-Rabbit Alexa 555. 
		For Pax7+Laminin+MF20, add Goat Anti-Mouse IgG2b Alexa 647 in the mix.</li>
		<li>Wash out the secondary antibodies with PBST at least 3x 5min.</li>
	</ol>
	</li>
	<li>DAPI (4&#39;,6-diamidino-2-phenylindole) counter-stain and mounting
	<ol>
		<li>Dilute DAPI (1:20,000) in PBST in the slide container, e.g., 10 &#181;L of DAPI in 200 mL of PBST.</li>
		<li>Stain the slides in the DAPI container for 3 min.</li>
		<li>Wash out the DAPI with PBST for at least 5x 5 min.</li>
		<li>Mount the slides with mounting medium. Cover the slides with coverslips. 
		CAUTION: DAPI is toxic and hazardous; it should be handled with care and disposed in the hazardous waste bottle.</li>
	</ol>
	</li>
</ol>
6. Fluorescent Microscopy
NOTE: The immunofluorescent staining protocol reported above will allow SCs visualization with either a wide-field fluorescent or confocal microscope. It may be initially challenging to identify SCs. Here are a few recommended steps that may be of help.
<ol>
	<li>Use the DAPI channel and low magnification objectives to visualize the sections on the slides.</li>
	<li>Switch the magnification from low to high. To observe SCs, at least a 20X objective is required).</li>
	<li>Use the dual filter cube for 488 (FITC)/555 (Rhodamine) to observe both Pax7 and Laminin signals at the same time to identify SCs. Under this setting, the signal of Pax7 staining has a better contrast to the background noise.</li>
	<li>Quickly alternate the channel from FITC to DAPI to correctly identify SCs. All SCs should be Pax7/DAPI-positive.</li>
</ol>

<ol>
	<li>Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+and+cell+lineages+that+underlie+the+formation+of+skeletal+muscle.">Gene regulatory networks and cell lineages that underlie the formation of skeletal muscle.</a> Proc Natl Acad Sci U S A. 114 (23), 5830-5837 (2017).</li><li>Buckingham, M., Relaix, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX3+and+PAX7+as+upstream+regulators+of+myogenesis.">PAX3 and PAX7 as upstream regulators of myogenesis.</a> Semin Cell Dev Biol. 44, 115-125 (2015).</li><li>Relaix, F., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+insect+eye+to+vertebrate+muscle:+redeployment+of+a+regulatory+network.">From insect eye to vertebrate muscle: redeployment of a regulatory network.</a> Genes Dev. 13 (24), 3171-3178 (1999).</li><li>Chang, N. C., Chevalier, F. P., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+Cells+in+Muscular+Dystrophy+-+Lost+in+Polarity.">Satellite Cells in Muscular Dystrophy - Lost in Polarity.</a> Trends Mol Med. 22 (6), 479-496 (2016).</li><li>Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cell+of+skeletal+muscle+fibers.">Satellite cell of skeletal muscle fibers.</a> J Biophys Biochem Cytol. 9, 493-495 (1961).</li><li>Kuang, S., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+biology+of+satellite+cells+and+their+therapeutic+potential.">The emerging biology of satellite cells and their therapeutic potential.</a> Trends Mol Med. 14 (2), 82-91 (2008).</li><li>Wang, Y. X., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+the+engines+of+muscle+repair.">Satellite cells, the engines of muscle repair.</a> Nat Rev Mol Cell Biol. 13 (2), 127-133 (2011).</li><li>Rinaldi, F., Perlingeiro, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+skeletal+muscle+regeneration:+therapeutic+potential+and+roadblocks.">Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks.</a> Transl Res. 163 (4), 409-417 (2014).</li><li>Le Grand, F., Rudnicki, M. A. <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+satellite+cells+and+adult+myogenesis.">Skeletal muscle satellite cells and adult myogenesis.</a> Curr Opin Cell Biol. 19 (6), 628-633 (2007).</li><li>Giordani, L., Puri, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+control+of+skeletal+muscle+regeneration:+Integrating+genetic+determinants+and+environmental+changes.">Epigenetic control of skeletal muscle regeneration: Integrating genetic determinants and environmental changes.</a> FEBS J. 280 (17), 4014-4025 (2013).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nat Protoc. 10 (10), 1612-1624 (2015).</li><li>Feng, X., Adiarte, E. G., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+acts+directly+on+the+zebrafish+dermomyotome+to+promote+myogenic+differentiation.">Hedgehog acts directly on the zebrafish dermomyotome to promote myogenic differentiation.</a> Dev Biol. 300 (2), 736-746 (2006).</li><li>Juan, A. H., 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=Polycomb+EZH2+controls+self-renewal+and+safeguards+the+transcriptional+identity+of+skeletal+muscle+stem+cells.">Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.</a> Genes Dev. 25 (8), 789-794 (2011).</li><li>Jackson, K. A., Snyder, D. S., Goodell, M. 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The authors have nothing to disclose.

The above protocol was based on a method of Pax7/MF20 staining on zebrafish skeletal muscle12. The solutions used and blocking steps are identical or similar. The antibodies used are identical. The adjusted steps were based on the features of mouse muscle tissue and SCs. First, Laminin antibody was added in the mix to help visualize and confirm the position of SCs. It was particularly helpful to count the number of SCs under the microscope when using the dual filter cube of 488/555; this greatly e...

Skeletal muscle is composed of multinucleated muscle cells, called myotubes, organized in myofibers, which generate force and movements through contraction. Most skeletal muscles, except for some craniofacial muscles, are derived from a temporary embryonic structure called a somite1. Myogenic precursor cells delaminate from the epithelial somite to become myoblasts. Myoblasts further differentiate into myocytes that fuse to become myotubes to form multi-nucleated myofibers. The above process is called myogenesis and is characterized by temporally-regulated control of gene expression. Myogenic precursors express Pax3 and Pax7, whereas myoblasts express MyoD and/or Myf5 and myocytes express myogenin and myosins2,3. Muscle growth is a process in which myofibers become larger by incorporating more myonuclei into existing fibers (hyperplasia) and by an increase in muscle fiber size (hypertrophy)4. During muscle growth, there is a sustainable source of myogenic cells that have stem cell properties in that they can differentiate and self-renew. These cells are termed satellite cells based on their physical location between the sarcolemma (cell membrane of the myofiber) and the basal lamina5. SCs vigorously contribute to muscle growth in the juvenile stage (the first 2-3 weeks of postnatal mice), but become quiescent in resting adult muscle6. Remarkably, they can be re-activated in response to muscle injures and differentiate into new muscle cells to repair the damaged muscle7.
The stem cell properties make the study of SCs relevant for both basic muscle biology and therapies of muscle diseases8. As a result, it has been an area of intense investigation in the past decades. A tremendous progress has been made in dissecting the genetics and epigenetics of SCs9,10. Techniques involved in isolating and identifying SCs in situ were developed and optimized along the way11. Immunofluorescent staining allows the identification of SCs through the use of specific antibodies, including that for Pax7. However, the scarcity and small size of the SCs combined with a strong auto-fluorescence of adult skeletal muscle tissue render the visualization challenging. Here, we describe an immunofluorescent staining protocol optimized for mouse muscle tissue for Pax7 and based on an existing method for zebrafish muscle12. In addition, a Laminin antibody labeled with a distinct fluorophore is employed to identify the basal lamina under which the SCs are located. This protocol consistently allows the visualization of Pax7-positive SCs and myogenic precursors under all tested physiological conditions and developmental stages.

<table>
	<tbody>
		<tr>
			<td>methylbutane</td>
			<td>Sigma-Aldrich</td>
			<td>M32631</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature (O.C.T.) compound</td>
			<td>Electron Microscopy Sciences</td>
			<td>62550-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco, Themo Fisher</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>16% PFA</td>
			<td>TED PELLA</td>
			<td>50-00-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Thermo Fisher</td>
			<td>0 1-6201</td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td>115-007-003</td>
			<td></td>
		</tr>
		<tr>
			<td>20x Citrate Buffer</td>
			<td>Thermo Fisher</td>
			<td>00 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pax7 mono-clonal mouse antibody (IgG1) (supernatant)</td>
			<td>Developmental Study Hybridoma Bank</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin polyclonal rabbit antibody</td>
			<td>Sigma-Aldrich</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr>
			<td>MF20 mono-clonal mouse antibody (IgG2b) (supernatant)</td>
			<td>Developmental Study Hybridoma Bank</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A-21121</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG2b cross-absorbed secondary antibody, Alexa Fluor 647</td>
			<td>Thermo Fisher</td>
			<td>A-21242</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555</td>
			<td>Thermo Fisher</td>
			<td>A32732</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1860 cryostat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DM6000 wide-field fluorescent microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMR wide-field fluorescent microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM510 confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM780 confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cuisinart electronic pressure cooker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of skeletal muscle satellite cells by immunofluorescence with pax7 and laminin antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

Following the above steps, SCs can be successfully visualized in adult resting muscle sections under a fluorescent microscope (Figure 1). Although the adult muscle tissue has strong auto-fluorescence in certain type of fibers, the bright Alexa series dyes can overcome the background noise and the signal stands out (Figure 1A, B; arrow heads). The two-photon confocal microscopy captures a relatively cleaner image ...

Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

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20.8K Views.  National Institutes of Health. The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Video: Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...