Determination of Muscle Fiber Type in Rodents

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Skeletal muscles consist of muscle fibers that can differ in both composition and functional characteristics. These three types of muscle fibers, broadly categorized as slow, fast IIa, and fast IIb muscle fibers, express characteristic myosin heavy chain proteins and have different metabolic and enzymatic activities, which can be used as surrogate markers to identify the different fiber types. Pathological changes affecting the muscle, such as denervation, muscle disuse, and atrophy not only manifest on a functional level, but also as marked changes in the composition of muscle fiber type of individual muscles. In this unit we describe three methods for histological identification of slow/type I, fast fatigue resistant/type IIa, and fast fatigable/type IIb fibers by staining for either myosin ATPases or oxidative enzyme capacity (succinate dehydrogenase, SDH)?or, alternatively, immunostaining for specific myosin heavy chain isoforms in muscles of mouse hindlimbs. Curr. Protoc. Mouse Biol. 2:231?243 © 2012 by John Wiley & Sons, Inc.

Keywords: SDH; ATPase; myosin; oxidative enzymes; muscle fiber type

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Introduction
Basic Protocol 1: Myofibrillar ATPase Staining of Fresh Muscle Sections
Basic Protocol 2: Histological Staining for Succinate Dehydrogenase (SDH)
Alternate Protocol 1: Fiber Typing of Mouse Skeletal Muscle Using Antibodies Against Specific Myosin Isoforms
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Myofibrillar ATPase Staining of Fresh Muscle Sections

Materials

Sodium acetate, trihydrate
0.1 M and 0.1426 M sodium barbital
Pre‐incubation solutions, pH 10.2, 4.5, and 4.2 (see Table 11.2.2900 )
Snap‐frozen muscle
Muscle sections cut transversely at 12‐ to 16‐µm on a cryostat (sections can be stored at −20°C until processed for staining)
Distilled water
Adenosine triphosphate (ATP), disodium salt
0.18 M calcium chloride (CaCl₂ )
1% (w/v) CaCl₂ solution
2% (w/v) cobalt chloride solution
5 mM sodium barbital (prepared as a 1:20 dilution from 0.1 M sodium barbital)
2% (v/v) ammonium sulfide solution (prepare in a fume hood fresh, immediately before use)
Ethanol solutions: 50%, 70%, 80%, 95%
Tissue clearing solution (Xylene or Histo‐Clear; National Diagnostics)
DPX mounting medium (Leica)

Staining racks
Staining jars
Forceps

NOTE: Snap‐frozen muscle: muscle mounted vertically on specimen block and covered with tissue embedding medium, e.g., OCT Compound (Tissue‐Tek), snap frozen in melting isopentane (−160°C) and stored at 80°C until cut.

Table 1.2.1 MaterialsQuick Reference for Pre‐incubation Solutions for ATPase Staining

Starting reagents and concentrations	Pre‐incubation solution volumes (ml) a
Reagent	[Reagent], M	pH 10.2	pH 4.5	pH 4.2
Sodium barbital	0.10	4.0	0.0	0.0
Calcium chloride	0.18	4.0	0.0	0.0
Barbital acetate solution b	0.14	0.0	5.0	5.0
HCl	1.00	0.0	0.0	10.0
HCl	0.10	0.0	10.0	0.0
Deionized H₂ O	—	11.0	4.0	4.0
Use the following for pH adjustment:		0.1 N NaOH	1.0 N HCl	0.1 N HCl

^a Note that reagent volumes are based on a final volume (after pH adjustment) of 20 ml; 1 ml of volume has therefore been reserved for pH adjustment.

^b ∼0.14 M barbital acetate solution (made by adding 0.97 g sodium acetate to 50 ml of 0.1426 M sodium barbital).

Basic Protocol 2: Histological Staining for Succinate Dehydrogenase (SDH)

Materials

SDH staining solution (Table 11.2.2900 )
Muscle biopsies, transversely cut at 12 µM
0.9% saline
70% and 90% acetone
100% ethanol
Tissue clearing solution (e.g., Histo‐Clear; National Diagnostics)

DPX mounting medium (Leica)

Table 1.2.2 MaterialsSDH Working Solution Quick Reference

Stock solution	[Stock solution], mM	10 ml	40 ml c
Phosphate buffer, pH 7.6	100.0	8.20	32.80
Na‐succinate buffer	1000.0	0.50	2.00
Nitroblue tetrazolium	15.0	1.00	4.00
KCN	100.0	0.10	0.40
Phenazine methosulfate d	10.0	0.20	0.80

^c For this protocol, make 40 ml SDH working solution.

^d Phenazine methosulfate is very sensitive to light. Make this solution fresh and store protected from all light.

Alternate Protocol 1: Fiber Typing of Mouse Skeletal Muscle Using Antibodies Against Specific Myosin Isoforms

Materials

Dissected muscle tissue, immediately snap frozen in melting isopentane and kept at −80°C
Blocks for cryosection prepared using of molds of appropriate size
Muscle sections cut transversely at 10 µm on a cryostat at −20°C and placed on gelatin‐coated slides
Blocking solution: 4% bovine serum albumin (BSA) in TBS with 0.10% Triton X‐100 (TBST)
Fab fragments (Jackson ImmunoResearch)
1× phosphate‐buffered saline (PBS) or PBS tablet (Sigma) dissolved in 200 ml deionized H₂ O
Primary antibodies: monoclonal or polyclonal antibody against a specific myosin isoform
Secondary antibodies
Mounting solution (e.g., Vectashield from Vector Laboratories or Fluoromount from Sigma)
Clear nail polish or other hard sealant (e.g., covergrip from Biotium)

Hydrophobic barrier pen (e.g., ImmEdge Pen from Vector laboratories)
Histology jars
Forceps
Plastic pipets
Plastic box (to hold several slides in humid environment)
Whatman filter papers, 1.5‐mm thick (Whatman, cat. no. GB005)
Coverslips
Appropriate microscope

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Figures

Figure 1. ATPase staining in mouse hindlimb soleus (A and C ) and TA muscles (B and D ). A and B shows ATPase staining in alkaline pre‐incubation conditions. It can be seen that in the soleus very dark and intermediate staining is predominant, indicating the presence of both fast and slow fibers, whereas in the TA there is an almost uniform presence of partly stained fast fibers. In contrast, pre‐incubation of muscles using acidic conditions (here pH 4.2 pre‐incubated sections are shown) results in slow type 1 fibers being very dark and fast fibers staining very lightly. Both stainings indicate that the soleus muscle in the mouse is a mixed muscle containing fast and slow fibers in almost equal proportions, whereas the TA muscle in the mouse is a predominantly fast one. Scale bar: 50 µm

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Figure 2. SDH staining in mouse hindlimb muscles. (A ) Shows SDH staining in mouse TA, EDL, and soleus muscles under low magnification. Note the mosaic pattern of darkly, lightly and intermediately stained fibers in EDL, the predominance of dark fibers in the soleus muscle, and the characteristic distribution of dark fibers (towards the center of the muscle) and light fibers (in the outside edges of the muscle) in TA muscles. (B ) Shows a large magnification image from the TA muscle outlined in A. A mosaic pattern of darkly stained, predominantly oxidative fibers (asterisk), very lightly stained glycolytic fibers (black arrow), and some intermediate fibers that display a staining that is intermediate between oxidative and glycolytic fibers (white square). Scale bars: (A) 500 µm; (B) 50 µm

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Figure 3. A comparison of the performance of two antibodies against MyHC‐I. Cross‐sections from the soleus muscle, a slow muscle of the hindlimb, were stained using rabbit polyclonal anti‐MYH7 (Sigma, cat. no. HPA001239) or mouse monoclonal BA‐D5. A typical checkerboard pattern is obtained with both antibodies. Dark unstained fibers are type IIa fibers, which do not express MyHC‐I.

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Figure 4. Example of fiber type change in the soleus muscle. (A ) This is a cross‐section of the soleus muscle from an 8‐month‐old homozygous ky mutant (Blanco et al., ), stained with BA‐D5 (anti‐MyHC‐I). (B ) Shows an age‐matched control. Note that the typical checkerboard pattern of the soleus muscle is lost in the ky sample, as in old ky mice all fibers become type I, i.e., express MyHC‐I.

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Figure 5. Example of identification of type IIa and type IIb fibers using monoclonal antibodies against MyHC‐IIa (SC‐71; green) and MyHC‐IIb (BF‐F3; red). Note that the soleus muscle do not express type IIb fibers (no red fibers present), while the gastrocnemius predominantly express type IIb fibers (red), as well as IIa (green) and IId (not stained) fibers.

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Videos

Literature Cited

Literature Cited
	Blanco, G., Coulton, G.R., Biggin, A., Grainge, C., Moss, J., Barrett, M., Berquin, A., Marechal, G., Skynner, M., Van, M.P., Nikitopoulou, A., Kraus, M., Ponting, C.P., Mason, R.M., and Brown, S.D. 2001. The kyphoscoliosis (ky) mouse is deficient in hypertrophic responses and is caused by a mutation in a novel muscle‐specific protein 1. Hum. Mol. Genet. 10:9‐16.
	Brooke, C.H., Allen, D.L., and Leinwand, L.A. 2011. IIb or not IIb? Regulation of myosin heavy chain gene expression in mice and me. Skeletal Muscle 1:5 doi:10.1186/2044‐5040‐1‐5.
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Determination of Muscle Fiber Type in Rodents

Abstract

Table of Contents

Materials

Basic Protocol 1: Myofibrillar ATPase Staining of Fresh Muscle Sections

Basic Protocol 2: Histological Staining for Succinate Dehydrogenase (SDH)

Alternate Protocol 1: Fiber Typing of Mouse Skeletal Muscle Using Antibodies Against Specific Myosin Isoforms

Figures

Videos

Literature Cited