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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

Published: November 13th, 2013

DOI:

10.3791/50259

1Departments of Pediatrics and Neurology, University of Michigan

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 μm) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Skeletal muscle is a highly specialized tissue responsible for generating the contractile forces necessary for motility. Contraction is initiated through a process known as excitation-contraction (EC) coupling that converts electric signals to calcium release from intracellular stores1,2 . Intracellular calcium release activates the sarcomere to shorten and generate force. The many specific components of the molecular machinery responsible for mediating neuromuscular junction transmission3, EC coupling4,5 , and actin-myosin dependent contractions6 continue to be the ongoing subject of intense research. In addition, proteins ....

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1. Preparation of Poly-L-Lysine Coated Coverslips (Time: 1 hr)

Coating coverslips allows for rapid myofiber settling and adhesion. This may be performed during the dissociation step of the myofiber isolation (step 2 below).

  1. Cut and place Parafilm on the bottom of a 60 mm Petri dish (any brand).
  2. Place microscope cover glass slips (12 mm diameter) on Parafilm in a 60 mm tissue culture dish or place them individually in single wells of 24-well plates.

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Fluorescent immunolabeling of myofibers (Figure 2)

Images showing expected fluorescent labeling pattern from myofibers immunostained after successful isolation and plating. The myofibers have been labeled with either anti-ryanodine receptor (1:100) (Figure 2A) or anti-α-actinin (1:100) (Figure 2B) antibodies, and reveal immunostaining of the triad and the Z-band respectively. Secondary antibodies used were Alexa Fluor 555 (1:500.......

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Zebrafish are a powerful vertebrate model system for studying muscle development and function in vivo25,27,28 . They have also emerged as a valuable asset for modeling human muscle diseases14,15,20,29 . While great strides have been taken to advance the use and application of zebrafish for the study of muscle function and muscle disease, there is a constant critical need to develop tools that will allow more in depth analysis that compliments the genetic, morphologic, behavio.......

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The authors wish to thank the members of the Dowling lab (Aaron Reifler, Trent Waugh, Angela Busta, and William Telfer) that contributed to the development of the technique and to the production of the manuscript. This work was funded by the Taubman Institute, the Department of Pediatrics at the University of Michigan, and in part from grants from the Muscular Dystrophy Association (JJD MDA186999) and the National Institutes of Health (JJD 1K08AR054835).

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
24-well culture plate Corning 3524
10x PBS Invitrogen Gibco 70011
CO2 Independent medium Invitrogen Gibco 18045
Collagenase Type II Worthington Biochemical LS004186 Lot 41H12764
Collagenase Type IV Worthington Biochemical L5004188
8% Paraformaldehyde Electron Microscopy Sciences 157-8
Methanol Sigma 322415
Triton X-100 Sigma X100
BSA Sigma A2153
Sheep serum Sigma S3772
Goat serum Sigma G9023
Glass coverslips Fischerbrand 12-545-82 12CIR-1D
Poly-L-Lysine Sigma P4707
Pronase Sigma P5147
40 μm Filter BD Biosciences 352340
70 μm Filter BD Biosciences 352350
Prolong Gold antifade reagent Invitrogen P36931
Anti-α-Actinin antibody Sigma A5044
Anti-RYR antibody Abcam 34C
Alexa Fluor antibody Invitrogen A-21425
TWEEN 20 Sigma P1379
60 mm Petri dish Fischerbrand 0875713A
Poly-L-Ornithine Sigma P4957
Microscope slide Fischerbrand 12-550-15
Caffeine Sigma C0750

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