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.     

An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.     

Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.     

Cytochemical and autoradiographic studies of the localization and turnover of chromatin and nucleolar associated phospholipids in plant and animal tissues

The localization of chromatin-associated phospholipids has been demonstrated on chromosomes and on chromatin of interphase nuclei by cytochemical methods either in plant and in animal tissues. Three methods of fixation are suggested which can be combined which two cytochemical methods for phospholipids detection. An additional method is represented by autoradiographic technique after incorporation of a radioactive precursor such as [3H] ethanolamine. This method has been used with good results in nuclei of lateral root apices of Vicia faba on 1 micron thick section, thus avoiding any possible contamination by nuclear membrane. In these nuclei the phospholipids appear associated with the chromatin and more intensely with the nucleolar regions. The positivity of the cytochemical reaction disappears after extraction of fixed tissue with acidified methanol chloroform and after treatment of unfixed sections with phospholipase D. The use of phospholipid precursor has allowed the study of chromatin-phospholipids synthesis in root apices of Vicia faba with respect to timings of the cell cycle. The results show that there is a strong case for a pattern of chromatin phospholipid synthesis which operates during S phase. Concerning the role of phospholipids it is suggested that they may be linked to acidic protein and may have a structural function, particularly on the nucleoli.     

Iodopolatinate visualization of phospholipids in rat incisor predentine and dentine,compared with malachite green aldehyde

The iodoplatinate (IP) reaction, a selective method for visualization of phospholipids, was applied to the predentine and dentine of rat incisors and compared with malachite green aldehyde (MG) fixation/staining. Spot tests indicated (1) that IP specifically stains phospholipids, but not amino acids, displaying as do phospholipids, quaternary ammonium groups; and (2) phosphatidylserine and sphingomyelin were also stained by MGA. Although this reagent is known to interact with phosphorus, phosphoproteins remained unstained. In the rat incisor, an IP-positive network including granules and thin filaments was seen in predentine in the inter-collagen spaces, in many cases closely associated with collagen fibres and their periodic striations. In dentine, positively stained needle-like structures were located along individual collagen fibres, or at the surface of groups of collagen fibres. This staining pattern was unchanged on sections of material pretreated with acetone, whereas the staining was abolished or markedly reduced when the samples were treated either with chloroform/methanol or phospholipase C prior to the IP reaction. Pretreatment of the samples with hyaluronidase promoted subsequent diffusion of the staining. A very similar staining pattern was observed with MGA, in accordance with earlier reports. The present findings validate the histochemical results reported previously on the distribution and potential role(s) of phospholipids in dentine biomineralization.     

A specific histochemical method for phospholipids and sulpholipids

Synopsis A new histochemical method is described for the polychromatic staining and identification of phospholipids and sulpholipids in tissue sections. It is an adaptation of a chromatographic method described previously (Kennedy & Collier, 1962). Sections are stained in a mixture of Diamond Sky Blue B, Diamond Cyanine R and formalin, followed by differentiation in 2-morpholinoethanol and bromine water, counterstaining in Cresyl Fast Violet and decolorization in lactic acid.The interpretation of the final colours in the test tissues was checked by extraction and chromatography of their lipid content.The staining of diverse animal and plant tissues is described and illustrated in colour.     

Biogenesis of mitochondrial membranes in Neurospora crassa during cellular differentiation: changes in oxidative phosphorylation and synthesis of mitochondrial phospholipids.

Changes in the capacity of mitochondria to carry out oxidative phosphorylation and in the rate of synthesis and incorporation of phospholipids into mitochondria were measured during the germination of conidiospores of Neurospora crassa. The competence of isolated mitochondria to carry out coupled respiration was very low during the first 3 h growth, but it increased rapidly, reaching maximal levels at 5 to 6 h growth. Changes in mitochondrial function were the same in cells grown in 2% sucrose- or 15% glucose-supplemented medium. The rate of synthesis of mitochondrial phospholipids was very low during the first 2 h growth and increased to maximal levels between 3 and 5 h. The rate of synthesis of mitochondrial phospholipids was approximately three times higher in cells grown in 15% glucose than in those grown in 2% sucrose. The maximal rate of synthesis of mitochondrial phospholipids occurred during spore germination and preceded attainment of full competence for oxidative phosphorylation. The lipid-rich condition of the mitochondrial resulting from the high rate of synthesis of phospholipids in glucose-grown cells is postulated to be related to the whorled inclusions observed in thin sections of Neurospora cells.     

Ultrastructural localization of anionic phospholipids in skeletal muscle plasma membrane

Polymyxin B was used as a probe to label anionic phospholipids in skeletal muscle plasma membrane. This antibiotic produces muscle surface membrane lesions that can be identified in both thin sections and freeze-fracture replicas. The membrane perturbations assumed a patchy distribution with a preferential localization at the level of the I band and A-I bands junction. Intramembraneous particles were also observed within the lesions. We consider the possibility that microdomains of anionic phospholipids in muscle plasma membrane may function in the binding of Ca++.     

Influence of specimen preparation on the identification of phospholipids by the phospholipase A2-gold method in mineralizing cartilage and bone

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A₂ (PLA₂)-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resiembedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA₂, which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mincralization suggests some hypotheses on the possible involvement of these molecules in the mineralphase deposition process.     

Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.     

Rapid-freezing and malachite green-acrolein-osmium tetroxide freeze-substitution fixation improve visualization of extracellular lipids in rat incisor pre-dentin and dentin

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.     

Biochemical aspects of the visual process. XXXVII. Evidence for lateral aggregation of rhodopsin molecules in phospholipase C-treated bovine photoreceptor membranes

Photoreceptor membranes derived from isolated bovine rod outer segments, are subjected to treatment with phospholipase C (Bacillus cereus). This results in varying degrees of hydrolysis of the membrane phospholipids into diglycerides and water soluble phosphate esters without loss of rhodopsin. Electron microscopic observations of thin sections and freeze-fractured preparations indicate extrusion of diglycerides from the membranes and their coalescence to lipid droplets, beginning at 20% hydrolysis of phospholipids. After 90% hydrolysis of phospholipids membranous structures are still present. The rhodopsin is located in these structures, presumably in the form of two-dimensional lateral aggregates. This explains the cross-fracturing of the membranous structures, regularly observed upon freeze-fracturing of the phospholipase-treated photoreceptor membranes.     

Radioiodination of naturally occurring phospholipids

A simple and rapid nonenzymatic method for radioiodination of phospholipids is described. It involves oxidation of Na125I with TlCl3 (or chloramine-T) in an aqueous medium, with subsequent exposure of the phospholipids, dissolved in chloroform/methanol, to the action of the oxidizing mixture. Purification of the radiolabelled phospholipids was effected by washing with sodium thiosulphate followed by thin-layer chromatography on silica gel. Specific radioactivity of 125I-labelled phosphatidylcholine was estimated to be about 10 muCi/mg phospholipid. The method is designed for radioiodination of various naturally occurring phospholipids.     

Quantitative histochemistry of myelin using Luxol Fast Blue MBS

Synopsis The amount of Luxol Fast Blue MBS in the band of Genarri was measured with two types of scanning microdensitometer and the optical density determined. The amount of stain measured was proportional to the section thickness employed, thus demonstrating that the dye has stoichiometric properties in tissue sections. Blocks of tissue treated with phospholipid solvents showed an increased uptake of stain, suggesting that phospholipids are not a primary substrate for the dye in myelin staining.The dye may, therefore, be used to quantify myelin in tissue sections.     

Nile Blue Histochemical Method for Phospholipids

The technic recommended is: Fix 6-12 hr. in 10% formalin containing 1% CaCl₂. Cut frozen sections without embedding or after gelatin or carbowax. Stain 90 min. at 60°C. in saturated aqueous Nile blue sulfate, 500 ml. plus 50 ml. of 0.5% H₂SO₄, boiled 2 hr. before use. Rinse in distilled water, and place in acetone heated to 50°C. Remove the acetone from the source of heat and allow the sections to remain 30 min. Differentiate in 5% acetic acid 30 min., rinse in distilled water, and refine the differentiation in 0.5% HCl for 3 min. Wash in several changes of distilled water and mount in glycerol jelly. Results: phospholipids - blue; everything else - unstained. Counterstaining nuclei with safranin is optional, but if done, it preferably precedes the Nile blue and is then differentiated by the acetic acid. The histochemical principles on which the method is based are as follows: (1) The calcium compounds of phospholipids combine with the oxazine form of Nile blue sulfate and survive subsequent treatment; (2) neutral lipids are dissolved out by acetone; (3) proteins and other interfering substances are destained by the acetic acid and hydrochloric acid baths.     

Demonstration of surfactant phospholipids in frozen sections of the lung

Saturated phospholipids are known to be the only surface active compounds present in the surfactant system of the lung. Using light microscopy, the identification in situ of pulmonary surfactant has always been hampered by the lack of satisfactory fixatives and dyes which act on saturated phospholipids fast enough to prevent the complete loss of surfactant in the solutions. In this study we adopted the tricomplex flocculation proposed by Elbers et al. (1965) to fix surfactant phospholipids on frozen sections obtained from human, pig and rat lungs. Small pieces of lung tissue were quickly frozen in freon 22 kept at -75 C.; eight micron sections were cut in a cryostat, air dried and immersed for 30s-5m in a 0.05 N Pb(NO3)2 + K3Fe (CN)6 solution in 10% formalin. Lead ions bound to the choline portion of phospholipid molecules were subsequently revealed in a 30 mM ammonium sulfide solution. This procedure delineates a dark brown filmy structure in the respiratory parenchyma, which is very loosely attached to the alveoli and appears to be related to lung surfactant. Preliminary lung lavage or pretreatment of sections with saline, aldehyde fixatives and several organic solvents, fully or partially abolish the stain.     

Sensitive analysis of ethanolamine- and serine-containing phosphoglycerides by high-performance liquid chromatography.

A highly sensitive method for the separation and quantitative measurement of phospholipids containing primary amino groups, such as phosphatidylethanolamine, phosphatidylserine and lysophosphatidylethanolamine, is described. The method involves a simple and quantitative derivative formation of the phospholipids containing amino groups to their u.v.-absorbing biphenylcarbonyl derivatives. These have molar extinction coefficients of about 23,000 at 268nm. The phospholipid derivatives are then separated and non-destructively determined by high-performance liquid chromatography. The amino phospholipids containing vinyl ether bonds (plasmalogens) can be determined separately from the diacyl- and alkylacyl-amino phospholipids. The lower limit of detection by high-performance liquid-chromatographic analysis of the phospholipid derivatives is about 10-13pmol or 0.3-0.4ng of phospholipid P. The quantitative range of derivative formation and analysis by high-performance liquid chromatography of the phospholipids containing amino groups was shown to be 10-500nmol. The method was shown to be applicable to the analysis of phospholipids containing amino groups in tissue samples.     

Phospholipids in plant and animal chromatin

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.     

Use of lead aspartate block staining in quantitative EM autoradiography of phospholipids: application to myelinating peripheral nerve

For quantitation of electron microscope (EM) autoradiographs, micrographs must contain clear images which are relatively free of heavy metal precipitates. Satisfactory contrast is usually obtained by staining individual ultra-thin sections with lead citrate. It was recently reported that sequential block staining of tissue with ferrocyanide-reduced osmium tetroxide and lead aspartate produced excellent contrast for EM autoradiography, with sections relatively free of lead precipitate. This protocol avoids the manipulation involved in staining individual ultra-thin sections. We have adapted this method to quantitative EM autoradiographic studies, primarily of phospholipid metabolism in peripheral nerve. We show that block staining with lead aspartate provides: (a) ultrastructural contrast of routinely high quality for myelinated peripheral nerve; (b) high (greater than 98%) retention of glycero-labeled lipid during dehydration and embedment; and (c) a distribution of de novo tritiated glycerol-labeled lipid in ultra-thin sections that is quantitatively identical to the distribution recorded for samples stained by the more laborious post-embedment method. During a 2-hr labeling period in vivo, tritiated glycerol is incorporated into phosphatidylcholine (44%), phosphatidylethanolamine (22%), other phospholipids (16%), and neutral lipids (15%). The analysis of grain distribution in developing sciatic nerve labeled for 2 hr with tritiated glycerol demonstrates that myelinating Schwann cells play the major role in synthesis of endoneurial lipids. Lipid synthesis in myelinated fibers is localized in perinuclear regions of Schwann cell cytoplasm. These regions lie external to compact myelin. Unmyelinated fibers and other endoneurial cells independently incorporate glycerol into lipids.     

Quantitative analysis of phospholipids by 31P-NMR

High-field 31P nuclear magnetic resonance spectroscopy was used to quantitate phospholipids in mixtures in organic solvents. The sample is dissolved in chloroform-methanol and analyzed at 161.7 MHz with decoupling of the protons. Signals were identified using authentic compounds, and their relative distribution was measured in mole percent. The method has good accuracy and reproducibility, and was used to analyze phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol, cardiolipin, and phosphatidic acid in egg lecithin. Four commercial egg phospholipids and the phospholipids from a total lipid extract of rat liver were analyzed. The method could be utilized to analyze phospholipids from other sources.