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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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

1. Euthanize the mouse in a CO₂ filled euthanasia chamber under the guidelines of the ACUC of the NIH. Perform cervical dislocation if needed.
2. Put the mouse face-up on a dissection pad (supine). Spray 70% et....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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