Name: identification of skeletal muscle satellite cells by immunofluorescence with pax7 and laminin antibodies
Uploaded: 2018-04-19T13:00:00
Description: Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

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
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	<li>Euthanize the mouse in a CO2 filled euthanasia chamber under the...

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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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