Determination of Muscle Fiber Type in Rodents

Bernadett Kalmar1, Gonzalo Blanco2, Linda Greensmith1

1 UCL Institute of Neurology, London, United Kingdom, 2 Department of Biology, University of York, York, United Kingdom
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110229
Online Posting Date:  September, 2012
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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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Table of Contents

  • 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 (CaCl 2)
  • 1% (w/v) CaCl 2 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 H 2O 11.0 4.0 4.0
Use the following for pH adjustment: 0.1 N NaOH 1.0 N HCl 0.1 N HCl

 aNote 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

     cFor this protocol, make 40 ml SDH working solution.
     dPhenazine 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 H 2O
  • 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

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.
   Brooke, M.H. and Kaiser, K.K. 1970. Three human myosin ATPase systems and their importance in muscle pathology. Neurology 20:404‐405.
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   Nachlas, M.M., Tsou, K.C., De Souza, E., Cheng, C.S., and Seligman, A.M. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p‐nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5:420‐436.
   Schiaffino, S., Gorza, L., Sartore, S., Saggin, L., Ausoni, S., Vianello, M., Gundersen, K., and Lømo, T. 1989. Three myosin heavy chain isoforms in type 2 skeletal muscle fibers. J. Musc. Res. Cell Motil. 10:197‐205.
   Schmalbruch, H. and Kamieniecka, Z. 1975. Histochemical fiber typing and staining intensity in cat and rat muscles. J. Histochem. Cytochem. 23:395‐401.
   Smerdu, V., Karsch‐Mizrachi, I., Campione, M., Leinwand, L., and Schiaffino, S. 1994. Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle. Am. J. Physiol. 267:C1723‐C1728.
   Smith, B. 1964. The Localization of enzymes within skeletal muscle fibers using the tetrazolium technique. J. Histochem. Cytochem. 12:847‐851.
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