Name: quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy
Uploaded: 2022-02-07T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy at JoVE.com

This work was supported by the Swedish Olympic Committee.

The present protocol using human biopsied skeletal muscle samples was approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20170198). Muscle biopsies were obtained through an incision in the skin from the vastus lateralis muscle using a Bergstr&#246;m needle with suction after local anesthesia was given subcutaneously (1-3 mL of Lidocaine 2% per incision). If isolated whole rat muscles were used, the animals were sacrificed by cervical dislocation before the muscle biopsies were ...

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The critical step of the method is the use of reduced osmium by potassium ferrocyanide during post-fixation. The selectivity of this modified fixative for glycogen detection cannot be fully explained by chemistry, but also includes experimental findings demonstrating no detection of such particles in tissues known to be free of glycogen or in the extracellular space11.
Critical parameters are the precision of the estimates and the fiber-to-fiber variation. By following ...

Glycogen particles are composed of branched polymers of glucose and various associated proteins1 and constitute an important fuel during high metabolic demands2. Although not widely recognized, glycogen particles also constitute a local fuel, where some subcellular processes preferentially utilize glycogen despite the availability of other and more long-lasting fuels as plasma glucose and fatty acids3,4.
The importance of storing glycogen as a subcellular specific localized fuel has been discussed in several reviews5,6 mainly based on some of the earliest documentations of the subcellular distribution of glycogen by transmission electron microscopy (TEM)7,8. The first studies used different protocols to increase the contrast of glycogen from histochemical staining techniques to negative and positive stainings9,10. An important methodological development was the refined post-fixation protocol with the potassium ferrocyanide-reduced osmium11,12,13,14, which significantly improved the contrast of glycogen particles. This refined protocol was not used in some of the pioneering work on exercise-induced glycogen depletion15 but was re-introduced by Graham and colleagues16,17.
Based on the 2-dimensional images, the subcellular distribution of glycogen is most often described as glycogen particles located in three pools: subsarcolemmal (just beneath the surface membrane), intermyofibrillar (between the myofibrils), or intramyofibrillar (within the myofibrils). However, glycogen particles could also be described as associated with, for example, sarcoplasmic reticulum7 or nuclei18. In addition to the subcellular distribution, the advantage of TEM-estimated glycogen content is also that quantification can be conducted at the single fiber level. This allows investigation of fiber-to-fiber variability and correlative analyses with fiber types and cellular components as mitochondria and lipid droplets.
Here, the protocol for the TEM-estimated fiber type-specific volumetric content of the three common subcellular pools of glycogen (subsarcolemmal, intermyofibrillar, and intramyofibrillar) in skeletal muscle fibers is described. The method has been applied to skeletal muscles from humans19, rats20, and mice21; as well as birds and fish22; and cardiomyocytes from rats23.

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			<td>Embedding 812 resin medium kit</td>
			<td>Taab</td>
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			<td>Glutaraldehyde solution 25%</td>
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			<td>ITEM</td>
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			<td>Leica</td>
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			<td>Olympus</td>
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			<td>Osmium tetroxide 4% solution</td>
			<td>Polysciences</td>
			<td>0972A</td>
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			<td>Philips CM 100 Transmission EM</td>
			<td>Philips</td>
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			<td>Potassium hexacyanoferrate (II) trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>455989-245G</td>
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quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

Using this protocol, glycogen particles appear black and distinct (Figures 1 and Figure 2). The normal values of glycogen are depicted in Figure 3. These data are based on a total of 362 fibers from 41 healthy young men as collected in different previous studies19,24,29,30,31</...

Watch this Scientific Journal Video about Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy at JoVE.com

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

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