PRESERVATION OF MYELIN LAMELLAR STRUCTURE IN THE ABSENCE OF LIPID : A Correlated Chemical and Morphological Study

The fine structure of myelin was studied in glutaraldehyde-fixed rat sciatic nerves depleted of lipid by acetone, chloroform:methanol (2:1 v/v), and chloroform:methanol:concentrated HCl (200:100:1, v/v/v). One portion of each of these nerves, plus the extracts, was saponified and analyzed by gas-liquid chromatography for fatty acids. The remainder of each nerve was stained in osmium tetroxide in CCl₄ (5g/100cc) and was embedded in Epon 812. Thin sections, examined in the electron microscope, revealed the preservation of myelin lamellar structure with a 170 A periodicity in nerves depleted of 98% of their lipids. Preservation of myelin lamellar structure depended on glutaraldehyde fixation and the introduction of osmium tetroxide in a nonpolar vehicle (CCl₄) after the lipids had been extracted. It is concluded that the periodic lamellar structure in electron micrographs of myelin depleted of lipid results from the complexing of osmium tetroxide, plus uranyl and lead stains, with protein.     

ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS

Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40 nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1-5 nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.     

Improvement of prolactin immunolabelling in osmium-fixed acrylic-embedded pituitary gland

Immunolabelling of prolactin (PRL) with protein A-colloidal gold complex and tissue fine structure were enhanced after postfixation of pituitary gland with osmium tetroxide and embedment in acrylic momers (LR White). Thin sections were treated with sodium metaperiodate before immunocytochemistry. An intense PRL labelling was detected in secretory granules, Golgi complexes and extracellular accumulation of the hormone. The use of osmium greatly improved the fine structure of the tissue and its stability during acrylic embedment.     

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy

Summary The usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.     

Extramembraneous particles and structural variations of tubular myelin figures in rat lung surfactant

Tubular myelin figures of pulmonary surfactant were examined by electron microscopy after fixation in glutaraldehyde and postfixation in an osmium tetroxide-ferrocyanide mixture. Bilayered membranes were seen as parallel arrays or as lattices with spacings varying from about 36 to 50 nm. This method also produced good visualization of drumstick-like particles, 5 nm in diameter and about 15 nm in length. The particles were regularly spaced at intervals of 16 nm in rows along the rectangular angles of myelin membranes. Depending on the size of the tubules the particles contacted each other in the center of the tubules at low diameters (tubular diameter less than 40 nm) and formed a continuous filamentous central core, or they were separated from one another (tubular diameter greater than 40 nm). In the latter case the central core had a hollow appearance. Based on further findings employing tannic acid, lipid extraction with 2,2-dimethoxypropane, and a ruthenium red-osmium tetroxide technique for the demonstration of polyanionic proteins it is suggested that these particles are protein in nature and that they are involved in the formation and maintenance of the structure of tubular myelin. A new concept of the ultrastructure of tubular myelin figures is proposed.     

An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.     

Studies of Osmium Deposits in the Carotid Body of the Cat

Double aldehyde fixed carotid bodies and small pieces of vagus nerve of cats were incubated in 3 mM copper sulfate and 0.5 mM potassium ferricyanide in 0.05 M acetate buffer (pH 5.6) for 30 minutes at room temperature. Several modifications of this procedure were also attempted. Tissues were then postosmicated with 2% unbuffered osmium tetroxide and heated to 50-55 C for ten minutes. Under the electron microscope carotid body cells exhibited fine osmium deposits within cisternae of endoplasmic reticulum, saccule and vesicles of Golgi complex, and cristae of mitochondria. Intense osmium precipitation was also noted in the mitochondria of nerve endings. In addition, much more intense, more conspicuous and more localized reaction wan noted in the intraperiod lines of the myelin sheath of nerves. Deposits here were rod-shaped, displaying considerable variation in length. These results are discussed in the light of previous findings on osmium deposits in various tissues. It was concluded that the osmium reaction is unspecific, and that histochemical methods employing hot osmium tetroxide to amplify enzymatic activities may therefore not be reliable.     

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV)

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.     

Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.     

The distribution of lipid in the cell structure: An improved method for the electron microscope

An improved partition method for visualizing lipid consists in fixing tissues in paraformaldehyde-glutaraldehyde followed by osmium tetroxide. Three progressive grades of lipid staining are then obtained: (i) by renewed osmium tetroxide alone, (ii) by partition in myrcene or farnesol solutions followed by renewed osmium, (iii) by saturated thymol in sucrose followed by partition and renewed osmium. No additional metallic stains are used. The thymol treatment in (iii) renders ‘masked’ lipid accessible to partition—the effect being regulated as required by time and temperature. Thymol used before the first osmium facilitates lipid extraction which provides a complementary test for lipid. The possibilities of the method have been demonstrated on sections of familiar tissues of insect (mainly Rhodnius, Hemiptera) and mammal (mouse). By and large the results support what is known already about the distribution of lipid in cells, but observations on lipid in muscle fibres, in the nucleolus and chromatin, in the cells of the adrenal cortex, in the lung and intestine suggest that the method might prove a source of new information.     

A rapid method for staining large chylomicrons

In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains.     

HISTOLOGICAL STAINING OF LIPIDS FOR THE LIGHT AND ELECTRON MICROSCOPE

1. It is generally agreed that the blackening of osmium tetroxide by unsaturated lipid is too unpredictable to demonstrate lipid in tissues.
2. At neutral pH osmium tetroxide combines with the double bonds in the lipoproteins of cellular membranes (mitochondria, etc.) and the deep colour reaction of ethyl gallate with this osmium provides good staining of lipid for the light microscope.
3. Osmium taken up by tissue proteins at neutral pH is only a small fraction of that taken up by the lipid. (After acid fixatives osmium tetroxide is a general protein stain.)
4. The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally fixed tissue most of the structural lipid is 'bound' and is not accessible to the dye.
5. Cautious treatment of fixed tissue with dilute sodium hypochlorite will unmask this lipid for viewing by the light microscope.
6. Direct fixation with neutral osmium tetroxide is an effective method for visualizing lipid for the electron microscope (as in the ethyl gallate method for the light microscope). But the poor penetration of osmium limits its use in this way.
7. After formol/glutaraldehyde fixation much of the lipid in the tissues is 'bound' and does not take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
8. The unmasked lipid can then be rendered more osmiophil by partition in a solution of the highly unsaturated terpene farnesol, thus increasing the uptake of osmium in a renewed application.
9. Some of the novel observations on tissue lipids made by these methods are reviewed.     

Filipin labelling and intramembrane particles on the membranes of early and later autophagic vacuoles in Ehrlich ascites cells

Cholesterol and intramembrane particle distribution on autophagic vacuole membranes was studied in Ehrlich ascites cells using filipin labelling and freeze-fracture electron microscopy. Unsaturated fatty acids were stained using imidazole-buffered osmium tetroxide. Autophagocytosis was induced with vinblastine, and early autophagic vacuoles were accumulated by lowering the ATP level in the cells with iodoacetate. Filipin labelling was observed in the limiting membranes of later, apparently hydrolase-containing autophagic vacuoles, whereas the most newly-formed, double-membrane limited vacuoles were not labelled. The limiting membranes of late, residual body-type vacuoles either showed patchy filipin-induced deformation or were completely smooth. Imidazole-buffered osmium tetroxide stained the membranes of newly-formed or developing autophagic vacuoles partly or entirely. The membranes of older vacuoles stained more weakly. Intramembrane particle density on the P-face of the outer limiting membranes of newly-formed autophagic vacuoles was similar to that on endoplasmic reticulum, and the density seemed to increase slightly later on. The size of the P-face particles increased when the vacuoles became older. The limiting membranes of late, residual body-type vacuoles were almost smooth. The inner limiting membranes and the membranes inside the autophagic were always almost particle-free. In conclusion, the amount of cholesterol, unsaturated fatty acids and protein in autophagic vacuole membranes changes during vacuole maturation.     

The molecular reorganization of lipid bilayers by osmium tetroxide. A spin-label study of orientation and restricted y-axis anisotropic motion in model membrane systems

Phospholipid dispersions spontaneously form oriented lamellar multilayers when dried onto glass slides. These oriented multilayers form useful model systems for studying the molecular dynamics of lipid bilayers. In order to examine the effects of osmium tetroxide on the orientation and motion of hydrocarbon chains in lipid bilayers, lecithin multilayers containing the spin label 3-doxyl-5α-cholestane (the 4′,4′-dimethyloxazolidine-N-oxyl derivative of 5α-cholestan-3-one) were prepared and examined by electron spin resonance spectroscopy. In egg lecithin multilayers at room temperature and 81% relative humidity the osmium tetroxide causes nearly complete loss of orientation and severe reduction of molecular motion. In contrast, the high degree of order in l-α-dipalmitoyl lecithin multilayers is not affected by exposure to osmium tetroxide vapors. Experiments are also reported on macroscopically disordered lecithin preparations, and the data support the conclusions drawn from the ordered lecithin multilayers that rotational mobility of the probe is severely reduced by fixation of the lipid chains.A simple mathematical model has been developed to account for the amplitude of the high-frequency (τ < 10^?8 sec) restricted y-axis anisotropic motion occurring in the bilayer plane. Since the y-axis is roughly parallel to the molecular axis of the rigid steroid spin label, this model enables quantitative comparisons of various degrees of restricted motion about the molecular axis.     

The reactions between formaldehyde,glutaraldehyde and osmium tetroxide,and their fixation effects on bovine serum albumin and on tissue blocks

Summary The reactions between osmium tetroxide and glutaraldehyde and formaldehyde were investigated. It was found that they react together to form intermediate products which then break down to form osmium black. Glutaraldehyde reacts much more rapidly with osmium tetroxide than formaldehyde. The rates of the reactions are increased by increasing the glutaraldehyde concentration or adding bovine serum albumin to the reaction mixture. The reaction rates increase with temperature. The mixtures of fixatives were also tried on tissues and the results paralleled the model experiments. The crosslinking of bovine serum albumin by osmium tetroxide, formaldehyde and glutaraldehyde singly and in mixtures was quantitatively assessed by viscosimetry, gel filtration and disc electrophoresis coupled with densitometry. The crosslinking of bovine serum albumin by pairs of fixatives was less than that produced by the most effective of the pair. After 5 min reaction osmium tetroxide was the most effective crosslinking agent according to viscosimetric experiments, but after one hour's reaction with bovine serum albumin, glutaraldehyde was revealed as the most effective crosslinking agent by gel filtration and electrophoresis.     

Density distribution of myelin fragments isolated from control and multiple sclerosis brains

Myelin from subcortical normal-appearing white matter of control and multiple sclerosis (MS) brains was isolated and subsequently subfractionated on discontinuous sucrose gradients. Three following myelin subfractions were obtained: light myelin (buoyant density ? 0.625 M), medium myelin (0.625 M > buoyant density ? 0.7 M), and heavy myelin (buoyant density > 0.7 M). The yield of total myelin (the sum of all three subfractions) recovered from MS specimens was about 30% lower than that from the white matter of the control brains. Furthermore, MS myelin was deficient in the light subfraction and was enriched in the heavy subfraction. No abnormality in lipid composition of MS subfractions was observed. On the other hand, myelin particles isolated from the MS tissue were depleted in basic protein. The results are interpreted as an evidence for a rather diffused pathological process in MS white matter.     

Isolation and Characterization of Two Types of Protein Bodies in the Rice Endosperm

Two types of proteinaceous particles were observed under the electron microscope in the starchy endosperm of rice seeds. One was spherical with lamellar structure (PB-I), while the other was stained homogeneously by osmium tetroxide and not lamellar structured (PB-II). Both types of proteinaceous particles were effectively condensed from the homogenate of developing rice endosperm by an aqueous polymer two-phase system using dextran-DEAE dextran-polyethylene glycol. Separation of both types was carried out by sucrose density gradient centrifugation. These proteinaceous particles were recovered at specific gravities of 1.27 and 1.29 for PB-I and PB-II, respectively. The protein composition of these particles and their solubility fractionation were examined. Prolamin appeared in the PB-I fraction, whereas PB-II was rich in glutelin and globulin.     

31P nuclear magnetic resonance studies of the association of basic proteins with multilayers of diacyl phosphatidylserine

Lysozyme, cytochrome c, poly(L-lysine), myelin basic protein and ribonuclease were used to form multilayer dispersions containing about 50% protein (by weight) with bovine brain diacyl phosphatidylserine (PS). 31P nuclear magnetic resonance shift anisotropies, spin-spin (T2) and spin-lattice (T1) relaxation times for the lipid headgroup phosphorus were measured at 36.44 MHz. At pH 7.5, lysozyme, cytochrome c, poly(L-lysine) and ribonuclease were shown to increase the chemical shift anisotropy of PS by between 12-20%. Myelin basic protein altered the shape of the phosphate resonance, suggesting the presence of two lipid components, one of which had a modified headgroup conformation. The presence of cytochrome c led to the formation of a narrow spike at the isotropic shift position of the spectrum. Of the various proteins or peptides we have studied, only poly(L-lysine) and cytochrome c had any effect on the T1 of PS (1050 ms). Both caused a 20-30% decrease in T1 of the lamellar-phase phosphate peak. The narrow peak in the presence of cytochrome c had a very short T1 of 156 ms. The possibility is considered that the cytochrome Fe3+ contributes to the phosphate relaxation in this case. The effect of all proteins on the T2 of the phosphorus resonance was to cause an increase from the value for pure PS (1.6 ms) to between 2 and 5 ms. The results obtained with proteins are compared with the effects of small ions and intrinsic membrane proteins on the order and motion of the headgroups of lipids in bilayers.     

Localization of l-fucose in the root cap and slime of Zea mays L. utilizing fluorescent lectin conjugates and colloidal gold-lectin techniques

Ulex europaeus lectin (UEA) labelled with fluorescein isothiocyanate (FITC), rhodamine or colloidal gold, localized l-fucose in maize root cap cells and secreted root cap slime. Free-hand sections of maize root apices stained with FITC-UEA or rhodamine-UEA and examined by fluorescence microscopy yielded satisfactory results as long as the stains were freed of unconjugated dye, the sections treated with osmium tetroxide vapour to quench autofluorescence, and the samples incubated at 37°C. This resulted in successful labelling with a lower concentration of fluorochrome-lectin conjugate than reported by previous workers. Rhodamine-UEA was superior to FITC due to the lower primary fluorescence of the root tip observed under green light.Thin sections from glutaraldehyde fixed and Spurr's resin embedded maize root tips were treated with UEA bound to colloidal gold. Gold particles were found within sloughed cells and root cap cells, particularly concentrated over the Golgi complex, Golgi-derived vesicles and within the secretory slime products.     

A Mixture of Paraphenylenediamine and Imidazole: Its Effect on the Extraction of Lipid Droplets during Electron Microscopy Staining

To prevent extraction of lipids during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve microvesicular lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer (pH 9.0) containing 0.5% p-phenylenediamine and 0.5% imidazole immediately after postfixation. The stained sections of the immersed tissue slice showed blackened, well circumscribed lipid droplets similar to those in corresponding unstained sections. Moreover, highly contrasting features of the cellular architecture could be visualized with the double stained, as well as routinely prepared sections.