Encyclopedia of Experiments: Biological Techniques
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Transcript
Glycogen, a branched glucose polymer, is an energy reserve in subcellular intermyofibrillar, intramyofibrillar, and subsarcolemmal compartments within the human skeletal muscle.
To quantify subcellular glycogen distribution using transmission electron microscopy, TEM, obtain isolated human skeletal muscle tissue. Fix with glutaraldehyde to preserve tissue structure. Postfix the tissue in an osmium tetroxide and potassium ferrocyanide solution.
The reduced osmium-ferrocyanide complex binds to the hydroxyl groups in the glycogen, improving glycogen particles' contrast in the muscle. Additionally, membranes and sarcoplasmic reticulum — a membrane-bound organelle — get stained.
Dehydrate the tissue in increasing ethanol concentrations. Embed in resin, forming a solid block. Obtain ultra-thin sections with longitudinally-oriented muscle fibers; transfer onto TEM grids.
Immerse the grids in uranyl acetate, heavy metal solution. Uranyl ions bind to negatively-charged lipids and proteins, making them electron-dense. Stain with lead citrate solution.
Lead ions bind to the osmium-stained regions including the glycogen particles, enhancing the contrast. They also bind to uranyl acetate-reacted regions, intensifying electron opacity.
During TEM imaging, as electron beams pass through the tissue, stained electron-dense regions, including glycogen, scatter more electrons, while unstained regions transmit electrons.
In the high-contrast TEM image produced, glycogen particles appear distinct and dark; unstained regions appear lighter, facilitating visualization and quantification of subcellular glycogen distribution in muscle.
