Histopathological Evaluation of Skeletal Muscle with Specific Reference to Mouse Models of Muscular Dystrophy

Rebecca L. Terry1, Dominic J. Wells2

1 Department of Pathology and Pathogen Biology, Royal Veterinary College, North Mymms, Hertfordshire, 2 Neuromuscular Group, Department of Comparative Biomedical Sciences, Royal Veterinary College, London
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.19
Online Posting Date:  December, 2016
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The muscular dystrophies are a diverse group of degenerative diseases for which many mouse models are available. These models are frequently used to assess potential therapeutic interventions and histological evaluation of multiple muscles is an important part of this assessment. Histological evaluation is especially useful when combined with tests of muscle function. This unit describes a protocol for necropsy, processing, cryosectioning, and histopathological evaluation of murine skeletal muscles, which is applicable to both models of muscular dystrophy and other neuromuscular conditions. Key histopathological features of dystrophic muscle are discussed using the mdx mouse (a model of Duchenne muscular dystrophy) as an example. Optimal handling during dissection, processing and sectioning is vital to avoid artifacts that can confound or prevent future analyses. Muscles carefully processed using this protocol are suitable for further evaluation using immunohistochemistry, immunofluorescence, special histochemical stains, and immuoblotting. © 2016 by John Wiley & Sons, Inc.

Keywords: cryosectioning; dystrophy; histopathology; mdx; muscle; necropsy

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Table of Contents

  • Introduction
  • Basic Protocol 1: Skeletal Muscle Removal, Freezing, and Cryosectioning
  • Basic Protocol 2: H&E Staining
  • Basic Protocol 3: Analysis of H&E Histology
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Skeletal Muscle Removal, Freezing, and Cryosectioning

  • Mice: test strain and control mice of the same genetic background (e.g., mdx and C57Bl/10)
  • Isopentane (2‐methylbutane, C 5H 12)
  • Liquid nitrogen (LN2) in a Dewar
  • Sodium pentobarbital (overdose given intraperiotneally in mice), optional
  • 70% (v/v) ethanol
  • Optimal Cutting Temperature (OCT) Embedding compound (e.g., Bright Cryo‐m‐Bed, Bright Instruments)
  • Surgical instruments (Fine Science Tools, Interfocus; “Student‐quality” instruments are adequate for dissection) including:
    • Curved forceps (Dumont no.7; cat. no. 91197‐00)
    • Fine scissors (cat. no. 91460‐11)
    • Surgical scissors (cat. no. 91402‐12)
  • 22‐mm diameter cork discs (label in advance), packs of 100 (Bright Instruments)
  • Aluminum foil (label in advance)
  • Fine permanent marker
  • Animal transport boxes
  • Metal container for isopentane
  • Dissecting microscope (optional)
  • 22‐G needles
  • Long forceps (∼30‐cm; Fine Science Tools, cat. no. 11000‐30 or similar)
  • Tissue paper
  • Single‐edged razor blade
  • Storage box
  • −80°C freezer
  • Cryostat
  • Superfrost Plus glass slides (VWR International)
  • Slide racks
  • Paintbrushes, mixture of firm and soft bristles, 0.5‐cm width heads or similar
  • Microcentrifuge tubes for collecting intermediate sections

Basic Protocol 2: H&E Staining

  • Slides of frozen muscle sections, defrosted and air‐dried ( protocol 1)
  • Harris’ Hematoxylin (Sigma, cat. no. HHS16l): diluted 1:1 with tap water and filtered
  • Eosin (Gurr Certistain): diluted 1% (w/v) in tap water and filtered before use (shelf life 1 to 2 months)
  • Acid alcohol [70% industrial methylated spirit (IMS)/1% HCl made in fume hood]
  • IMS diluted to 70%, 80%, and 90% solutions with deionized water
  • Xylene
  • DPX mounting medium
  • Pencil
  • Coplin jars/slide racks and glass staining trays
  • Coverslips
  • Glass rods
  • Forceps
  • Tissue paper
  • Slide boxes
  • Brightfield microscope ± camera
NOTE: IMS is used as a cheaper alternative to ethanol.

Basic Protocol 3: Analysis of H&E Histology

  Additional Materials (also see protocol 2)
  • Slide boxes
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Literature Cited

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Internet Resources
  Standard operating procedures from the above site/are listed in the Literature Cited.
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