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J Vis Exp. 2014 Jul 15;(89). doi: 10.3791/51586.

Tissue triage and freezing for models of skeletal muscle disease.

Author information

  • 1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin.
  • 2Department of Physiology and Cell Biology, The Ohio State University.
  • 3Department of Human Nutrition, Foods and Exercise, Virginia Tech.
  • 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky.
  • 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School.
  • 6Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin; Cure Congenital Muscular Dystrophy.
  • 7Joshua Frase Foundation.
  • 8Department of Rehabilitation Medicine, University of Washington.
  • 9Department of Physiology, University of Arizona.
  • 10Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin; mlawlor@mcw.edu.

Abstract

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

PMID:
25078247
[PubMed - in process]
PMCID:
PMC4215994
Free PMC Article
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