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. 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+fiber-specific+green+autofluorescence:+potential+for+stem+cell+engraftment+artifacts.">Skeletal muscle fiber-specific green autofluorescence: potential for stem cell engraftment artifacts.</a> Stem Cells. 22 (2), 180-187 (2004).</li><li>Lepper, C., Conway, S. J., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+satellite+cells+and+embryonic+muscle+progenitors+have+distinct+genetic+requirements.">Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements.</a> Nature. 460 (7255), 627-631 (2009).</li></ol>

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 (IgG2&lt;sub&gt;b&lt;/sub&gt;) (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 IgG2&lt;sub&gt;b&lt;/sub&gt; 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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20.9K 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 ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

Genetics

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

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 ...

Biology

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

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

Summary

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Chapters in this video

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

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

An Efficient Protocol for CUT&RUN Analysis of FACS-Isolated Mouse Satellite Cells

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

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo