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.     

A histochemical method for the demonstration of unsaturated lipids

Summary A method has been developed for the histochemical demonstration of unsaturated lipids in light microscopy. It is a peracetic acid-thiocarbohydrazide-silver protein sequence followed by a physical development procedure. In the present study on paraffin and cryostat sections of liver, brain and ovary, unsaturated lipids were visualized as distinct reaction products coloured various shades of brown and black. The reaction products are easier to see and the method is more efficient than the peracetic acid-Schiff method.     

Bromine-Sudan Black: a general stain for lipids including free cholesterol

Synopsis Preliminary bromination (with bromine water) increases the intensity of staining of tissue lipids with Sudan Black and certain other dyes. The mechanism appears to be due to the formation of sudanophilic bromo-derivatives of cholesterol and to the retention of certain other lipids, notably phosphatidyl choline and free fatty acids, during staining. The advantage of the bromine-Sudan Black method is that all tissue lipids are stained, except saturated fatty acids, saturated triglycerides and perhaps saturated cholesterol esters. In practice, such lipids rarely, if ever, occur alone, and normally are admixed with their stainable unsaturated counterparts.     

Bioautography: A general method for the visualization of isozymes

A new method has been developed for visualization of isozymes which are difficult or impossible to detect with standard histochemical or autoradiographic methods. The principle of this method, bioautography, is the use of a microbial reagent to locate an enzyme after gel electrophoresis. When bioautography was compared to other staining procedures, the bioautographic method yielded identical results to those observed by the histochemical method for lactate dehydrogenase (LDH) or by the autoradiographic method for the adenine phosphoribosyltransferase (APRT). Using the bioautographic method, stains for enzymes which could not be visualized by any other procedure have been developed: argininosuccinate lyase and branched-chain aminotransferase. By employing appropriately genetically marked bacterial strains, it should be possible to develop new isozyme stains for a large number of unstudied isozymes.     

Chemical analysis of osmium tetroxide staining in adipose tissue using imaging ToF-SIMS

Osmium tetroxide (OsO₄) is a commonly used stain for unsaturated lipids in electron and optical microscopy of cells and tissues. In this work, the localization of osmium oxide and specific lipids was independently monitored in mouse adipose tissue by using time-of-flight secondary ion mass spectrometry with Bi cluster primary ions. Substance-specific ion images recorded after OsO₄ staining showed that unsaturated C18 fatty acids were colocalized with osmium oxide, corroborating the view that osmium tetroxide binds to unsaturated lipids. In contrast, saturated fatty acids (C14, C16 and C18) and also unsaturated C16 fatty acids show largely complementary localizations to osmium oxide. Furthermore, the distributions of saturated and unsaturated diglycerides are consistent with the specific binding of osmium oxide to unsaturated C18 fatty acids. The abundance of ions, characteristic of phospholipids and proteins, is strongly decreased as a result of the osmium staining, suggesting that a large fraction of these compounds are removed from the tissue during this step, while ions related to fatty acids, di- and triglycerides remain strong after osmium staining. Ethanol dehydration after osmium staining results in more homogeneous distributions of osmium oxide and unsaturated lipids. This work provides detailed insight into the specific binding of osmium oxide to different lipids.     

Studies in lipid histochemistry

Summary The histochemical value of fractionated histochromatographic examination of lipids according to Holczabek's (1969) method which gives very good results as regards chromatography, is limited by that phospholipids are not retained quantitatively in sites of their original localization in sections during the first developing in a mixture of petrolether:ether:acetic acid. The staining of phospholipids performed after the first developing therefore does not reveal the localization pattern of all phospholipids.     

A histochemical analysis of adrenal lipids in Ox and Sheep

Summary The adrenals of Ox and Sheep were analysed by various histochemical methods available for lipids. The cortex was found to be rich in choline containing phospholipids, unsaturated phospholipids and masked lipids compared with the medulla. Unsaturated lipids and free fatty acids were more abundant in the cortex. There was slightly more cholesterol and plasmalogen in the medulla than the cortex. Formaldehyde fixation apparently increased the relative number of carboxyl groups in the adrenaline storing cells. Osmium tetroxide reacted with catechol amines and differentiated between the adrenaline and noradrenaline storing cells in the ox medulla. The histochemical results are compared with previous biochemical findings on the nature and distribution of adrenal lipids.     

Changes of lipid composition in non-cultured and cultured porcine embryos

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.     

Study of the nature of lysosomes and of their acid phosphatase

Summary Treatment with M/2 CaCl₂ or fresh frozen sections of various organs of mice inhibits the subsequent histochemical demonstration of acid phosphatase by various procedures. This inhibition can be reversed by treatment with M/I NaCl. Fixation in cold formalin solution, or treatment with fat solvents weakens or abolishes this effect with the result that phosphatase staining is not markedly influenced by pretreatment with salts. The effect of surfactants in inhibiting acid phosphatase staining is diminished by fixation in formalin. Treatment with M/2 CaCl₂ of fresh frozen sections of mice organs partly inhibited the histochemical staining of phospholipids.The findings indicate that acid phosphatase molecules are bound to polar lipids and that under the influence of excess of Ca ions the lipoproteic molecules of acid phosphatase form droplets bounded by hydrophobic groups of the lipids. In such droplets the enzyme molecules are not available to act on substrates dissolved in the medium, and the droplets may be regarded as lysosomes. Under the influence of excess Na ions in the milieu the lipoprotein layer breaks into droplets bounded on the outside by the enzyme protein and the hydrophilic groups of the phospholipids. Such particles probably represent microsomes. The implications of these conclusions are discussed.A preliminary communication dealing with the present study has been published (in Hebrew) in the Harefuah (Wolman, 1964).     

Carbonic anhydrase in rat liver and rabbit skeletal muscle: further evidence for the specificity of the histochemical cobalt-phosphate method of Hansson

Rat liver and rabbit skeletal muscle were studied by Hansson's method for histochemical demonstration of carbonic anhydrase activity. In histochemical model experiments purified male rat liver carbonic anhydrase was much more resistant to acetazolamide than rat erythrocyte carbonic anhydrase. Male rat liver slices showed cytoplasmic staining, which was about 1000 times more resistant to acetazolamide and ethoxzolamide than that of female rat liver or erthyrocytes of either sex. Rabbit skeletal muscle slices showed staining at the sarcolemma of all fibers, whereas the staining of the sarcoplasm varied. The walls of capillaries situated within the muscle bundles were intensely stained. The sarcoplasmic staining of a certain number of fibers was at least 1000 times less sensitive to acetazolamide than the other staining. These findings, which are in good agreement with biochemical data, show that the sulfonamides inhibit histochemical staining in a specific way. This is strong evidence for the specificity of the method.     

Use of nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms

We describe the use of the fluorescent dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, as a general-purpose reagent for the rapid detection and quantitation of a wide variety of lipids and other hydrophobic compounds separated by thin-layer chromatography. After samples are applied to silica gel plates and chromatographed, the plate is briefly dipped into a nile red solution (8 micrograms/ml of methanol-water 80:20, v/v). Background fluorescence of nile red dye adsorbed to the silica gel is then preferentially destroyed by dipping the plate in a dilute aqueous solution of bleach. After drying, lipid bands are visualized under ultraviolet light. Reflectance fluorometry (Ex: 580 nm; Em: 640 nm) is utilized for in situ quantitative analysis of the fluorescence of the lipids on the nile red-stained plate. Neutral lipids, phospholipids, sphingolipids, and fatty acids can be examined, although the nile red fluorescence intensity varies significantly among the lipid classes. Also, staining is stronger for unsaturated lipids than for saturated lipids. The lower detection limit of the assay is 25-100 ng for cholesterol, cholesteryl esters, triacylglycerols, and phospholipids.     

A Comparison of Standard Lipid Staining Techniques Used in Electron Microscopic Studies of Mammalian Tissues

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylenediamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.     

A comparison of standard lipid staining techniques used in electron microscopic studies of mammalian tissues

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylene-diamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.     

A double protein A-gold-silver staining method for tissue antigens in light microscopy

Summary A double staining method has been developed for light microscopic immunohistochemistry. The technique consists of a sequence of protein A-gold-silver procedures, in which the reaction products are visualized in black and brown colours. The results obtained in the rat pancreatic islets and adenohyophysis, in combination with a variety of histochemical controls, substantiate both the specificity and reliability of this method. The double staining method is simple and economical, since the two differently coloured reaction products are obtained by a common mechanism of colouration (physical development) using a single probe, protein A-gold.     

Rapid Otan Method for Localizing Unsaturated Lipids in Lung Tissue Sections

The OTAN treatment, which is the only histochemical method available at present for the simultaneous localization of hydrophobic and hydrophilic unsaturated lipids in tissue sections, requires unduly long exposure to O₃O₄ and use of free-floating sections, which makes handling the sections difficult and often results in their loss or damage. Simple modifications using O₃O₄ treatment at 37 C and slide-mounted sections eliminate the practical drawbacks of the existing method and provide as good or better localization in less than one-eighth of the time. The modified method is applicable to fixed as well as fresh frozen tissues.     

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the omega 6 to omega 3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the omega 6 to omega 3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondrial membrane-associated enzymes.     

Differential Staining for Cellulosic and Modified Plant Cell Walls

A simple method to enhance the staining of cell wall components for fluorescence microscopy is described. In stems of Nicotiana tabacum and needles of Pinus eldarica lignin, the cuticle and unsaturated lipids are indicated by a purple-red fluorescence while pectocellulosic components fluorescc pale blue.     

Differential Staining of Two Subpopulations of Purkinje Neurons in Rat Cerebellum with Acid Dyes

We present a new method that stains differently two subpopulations of Purkinje cells in the adult rat. Deparaffinized sections of cerebella, fixed by perfusion with buffered glutaraldehyde or Bouin's fluid were stained with 0.5% light green in 50% ethanolf 10-30 min). The excess dye was removed with saturated aqueous picric acid (10-30 min). At this point some Purkinje cells appeared as lightly stained neurons, while others were strongly stained. Slides were immersed in 0.5% aqueous acid fuchsin for approximately 1 min until the lightly stained neurons acquired a red color. Following immersion in 1% phosphotungstic acid, slides were rapidly dehydrated in ethanol, passed to xylene and mounted in Canada balsam. Two subpopulations of Purkinje cells differing in their protein content in somata and proximal dendrites stained differentially by this method. They occurred in all coronal and sagittal sections and in patches or stripes. Their relative proportion varied from lobule to lobule. A second staining method used potassium permanganate as the sole staining reagent. The staining reagent can be used on sections previously stained with the acid dyes. Purkinje cells appeared as subsets of brownish to deep brown stained neurons, the latter ones corresponding to green stained cells in the dichromic method. The results obtained indicated that the subpopulations reflect real differences among individual neurons and are not artifacts. The technique holds promise for identifying and localizing subsets of Purkinje cells differing in their protein content under normal and experimental conditions and for their further characterization by combined staining and histochemical procedures.     

Complex hydrogenation/oxidation reactions of the water-soluble hydrogenation catalyst palladium di (sodium alizarinmonosulfonate) and details of homogeneous hydrogenation of lipids in isolated biomembranes and living cells.

Palladium di (sodium alizarinmonosulfonate) is a highly efficient catalyst for the hydrogenation of unsaturated fatty acids esterified in lipids of model or biological membranes, enabling the study of the relationship between function and the physical state of membranes. However, the catalyst shows a complex behavior in the action of molecular hydrogen and oxygen, giving rise to the formation of at least four products. Two of these are free radicals. Owing to this complexity, precise control of the reaction requires pretreatment of the catalyst. When partial hydrogenation of the palladium complex is followed by air oxidation, a catalyst solution is produced which is stable on air and maintains catalytic hydrogenation activity for several days. This form of the catalyst induces hydrogenation of unsaturated lipids with no induction period making a strict timing of the procedure possible. Of the several other factors affecting the outcome of membrane hydrogenations, one of the most important is the accessibility to the catalyst of particular membrane regions or lipid pools. Differences in accessibility may arise as a consequence of different local microviscosities or their change during hydrogenation, of the appearance of distinct liquid crystalline phases, and of strong protein-lipid interactions. Obviously, in case of whole-cell hydrogenations, the accessibility is influenced by the spatial separation of the organelles, as well.     

Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.