The Quantitative Retention of Cholesterol in Mouse Liver Prepared for Electron Microscopy by Fixation in a Digitonin-Containing Aldehyde Solution

Scallen, Terence J.; Dietert, Scott E.

doi:10.1083/jcb.40.3.802

Cited by 112 publications

(29 citation statements)

References 18 publications

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“…Rats were anesthetized as described above and perfused transcardially with 200 ml of 4O/o glutaraldehyde in a 0.05 M phosphate buffer (pH 7.4) over a 10-15 min interval. A 5 mm segment of the injected left sciatic nerve was excised from the region of the injection and immersion fixed for 24 hr in 4% glutaraldehyde containing 0.2% digitonin (to prevent the extraction of cholesterol in subsequent steps) (Napolitano and Scallen, 1969;Scallen and Dietert, 1969). Nerve segments were then postfixed in Dalton's chrome osmium for 1 hr, dehydrated rapidly in a series of ice-cold graded acetone solutions to maximize retention of lipids (Gould et al, 1987), and embedded in Spurr's resin.…”

Section: Morphological Methodsmentioning

confidence: 99%

Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study

et al. 1994

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Four weeks after labeling myelin lipids with an intraneural injection of 3H-acetate, sciatic nerves were crushed, and the distribution of radiolabeled myelin lipids was followed by autoradiography from 1 d to 10 weeks later. Just prior to crush, silver grains were localized to the myelin sheath. Three days after crush, axons were degenerating and myelin sheaths were breaking down; silver grains appeared over lipid droplets within Schwann cells, fibroblasts, and macrophages. One week after crush the basal-lamina-delimited Schwann-cell tubes (Bungner bands) contained myelin debris, and some tubes already contained regenerating axons. Schwann cells were often displaced to the periphery of the tubes by phagocytes containing heavily labeled myelin debris; extratubal macrophages within the endoneurium contained labeled lipid droplets but no myelin debris. Two weeks after nerve crush silver grains were associated with newly formed myelin around regenerating axons. Many extratubal endoneurial macrophages now contained labeled myelin debris and lipid droplets. By 3 weeks myelination of regenerating axons was advanced, and the myelin sheaths were well labeled. Extratubal macrophages had become the major labeled structure within the nerve because they contained large amounts of labeled myelin debris and lipid droplets. From 4 to 10 weeks after nerve crush the new myelin sheaths continued to thicken and to be well labeled. Debris- laden extratubal macrophages remained the major site of labeled material within the endoneurium. Our results confirm that there is reutilization of myelin cholesterol by Schwann cells to form new myelin, and indicate that some lipid catabolism takes place in Schwann cells and endoneurial fibroblasts prior to infiltration of the nerve by macrophages. However, most of the myelin debris is phagocytized by macrophages within 1–2 weeks following nerve injury. These debris-laden macrophages persist within the nerve for many weeks, indicating that much of the salvaged cholesterol is not reutilized for myelin regeneration.

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Section: Morphological Methodsmentioning

confidence: 99%

Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study

et al. 1994

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“…Digitonin is a steroid-based nonionic detergent derived from the seeds of Digitalis purpurea (265). It complexes with cholesterol and other unconjugated ␤-hydroxysteroids in membranes (102,339), resulting in an increase in the permeability of cell membranes to inorganic ions, metabolites, and enzymes without global changes in cell structures (109). Because the molar ratio of cholesterol to phospholipid in the plasma membrane is much greater than in organelle membranes, low concentrations of digitonin (10 -100 g/ml) selectively increase the permeability of plasma membrane, resulting in release of cytosolic indicator but retention of intracellular organelle indicator (186,298,440) (Fig.…”

Section: E Compartmentalizationmentioning

confidence: 99%

Measurement of Intracellular Calcium

Takahashi

Camacho

Lechleiter

et al. 1999

Physiological Reviews

671

511

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To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

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“…3-hydroxysterols present in the plasma membrane (7). Because the molar ratio of cholesterol to phospholipid in eukaryotic plasma membranes is severalfold or manyfold greater than in intracellular membranes (8), treatment of intact cells with low concentrations of digitonin can be expected to bring about a selective increase in the permeability of the plasma membrane.…”

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confidence: 99%

The cytoskeleton of digitonin-treated rat hepatocytes.

Fiskum¹,

Craig²,

Decker³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

219

137

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Treatment of isolated rat hepatocytes with low concentrations of digitonin increases the permeability of the plasma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentrations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes. Digitonin, a steroid glycoside, increases the permeability of different types of cells to inorganic ions (1, 2, *), metabolites (1-4, *), and enzymes (3,5,6). This effect is believed to be the result of the binding of digitonin to cholesterol and other 3-hydroxysterols present in the plasma membrane (7). Because the molar ratio of cholesterol to phospholipid in eukaryotic plasma membranes is severalfold or manyfold greater than in intracellular membranes (8), treatment of intact cells with low concentrations of digitonin can be expected to bring about a selective increase in the permeability of the plasma membrane. This property has been used in this and other laboratories to make measurements of transport activities by mitochondria (1, 2, 9, *) and endoplasmic reticulum (9) in cells in situ. Digitonin offers a major advantage over other commonly used agents, such as Triton, glycerol, or toluene, because the latter are relatively nonspecific in their effects on different types of cell membranes.Experiments described in this paper demonstrate another useful consequence of the treatment of cells with digitoninnamely, greatly enhanced electron microscopic visualization of certain intracellular structures, particularly the cytoskeletal network of filaments, which are normally difficult to observe due to the presence of dark-staining cytosolic protein. Such pretreatment of hepatocytes with low levels of digitonin causes no loss or impairment of the normal morphology of membrane-limited organelles, such as mitochondria, endoplasmic reticulum, and lysosomes. The enhanced visualization of intracellular structures as described here reveals some hitherto unreported associations of microfilaments and intermediate filaments within isolated normal hepatocytes, supplementing earlier reports on the role of microfilaments in maintenance of cell shape (10), in endocytosis (11), and in secretion of lipoproteins (10). MATERIALS AND METHODSIsolation...

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The Quantitative Retention of Cholesterol in Mouse Liver Prepared for Electron Microscopy by Fixation in a Digitonin-Containing Aldehyde Solution

Cited by 112 publications

References 18 publications

Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study

Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study

Measurement of Intracellular Calcium

The cytoskeleton of digitonin-treated rat hepatocytes.

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