Effect of resin use in the post-embedding procedure on immunoelectron microscopy of membranous antigens, with special reference to sensitivity

To investigate quantitatively the effect of resins on the sensitivity of immunoelectron microscopy of membranous antigen, ultra-thin sections of bovine epithelial tissue embedded in five different kinds of resins [JB-4 (JB4), LR Gold (LRG), Lowicryl K4M (K4M), Quetol 812 (Q812), and Spurr's (Spurr) resin] were labeled specifically with anti-desmosomal glycoprotein I(DGI) antibody followed by protein A-gold (PAG) conjugates. When we compared the labeling intensity expressed as the number of PAG particles per 500-nm length of the desmosomal region along the membrane, three hydrophilic resins (JB4, LRG, and K4M) showed much greater levels of labeling intensity than did epoxy resins (Q812 and Spurr), which had a negative value. The three hydrophilic resins showed only minor differences in their levels of labeling intensity. The intensity obtained with JB4, which was the highest of the three, was further increased by pretreatment of the ultra-thin sections with methyl methacrylate monomer (MM) for 5 min. On the basis of these results, wide applicability of this new technique for membranous antigens, which have been difficult to detect positively by any previously employed techniques, is suggested.     

Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria.

Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acid and wall components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetone-treated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells. Autoradiographs of specimens collected during freeze-substitution were scanned with an optical densitometer to provide an indication of freezing damage; the majority of label lost from freeze-substituted cells was a result of poor freezing to approximately one-half of the cell population, thus accounting for the relatively high levels of radiolabel detected in the processing fluids. These experiments revealed that gram-positive and gram-negative cells respond differently to freezing; these differences are discussed with reference to wall structure. It was apparent that the cells frozen first (ie., the first to contact the cryogen) retained the highest percentage of all radioisotopes, and the highest level of cellular infrastructure, indicative of better preservation. The preservation of these select cells was far superior to that obtained by more conventional techniques.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies

Summary A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies.

A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

Chemical Dehydration of Specimens with 2,2-Dimethoxypropane (DMP) for Paraffin Processing of Animal Tissues: Practical and Economic Advantages over Dehydration in Ethanol

Chemical dehydration can be accomplished using 2,2-dimethoxy-propane (DMP). In the presence of an acid catalyst, this liquid reacts with water generating methanol and acetone as products. Although DMP is more expensive per milliliter than ethanol and other solvents used for dehydration, it is an economical alternative because a much smaller volume is needed. Slow penetration of DMP was previously thought to restrict its use to tiny specimens, but we now show that pieces of tissue as thick as 2 cm are dehydrated by overnight immersion in acidified DMP. We also show that dehydration in acidified DMP does not impair the staining of UNA or other basophilic components of animal tissues. The temperature and concentrations of methanol and H⁺ in the chemical dehydrating agent are too low to produce histochemicaUy detectable methylation or nucleic acid extraction.     

Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.     

Better epoxy resin embedding for electron microscopy at low relative humidity.

In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used; impregnate tissues using the following solutions 1) 70% alcohol - 5 minutes, 2) 95% alcohol - 2 x 15 minutes, 3) absolute alcohol - 3 x 20 minutes, 4) acetone - 2 x 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 812, 39.4 g; DMP-30, 2.6 g) - 1 hour, 6) acetone-epoxy resin 1:3 - 1 hour, 7) epoxy resin - 1 hour; complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.     

The investigation of vinylcyclohexane dioxide as a polar dehydrant for the improved retention of lipids in the Spurr embedment

The use of vinylcyclohexane dioxide (VCD) as a polar dehydrant with subsequent embedment in Spurr was studied. The utilization of Epon 812 resin (E 812), hydroxyethyl methacrylate (HEM) and hydroxypropyl methacrylate (HPM) as polar dehydrants for Epon embedment were re-examined, and a polar Epon mix was introduced. The most effective dehydration sequence was: first 90%, then 95% VCD in water for 5 min. followed by two 20 min changes of 100% VCD. After 1 hr in equal quantities of VCD and Spurr mix, tissues were infiltrated with Spurr embedment (two 1 hr changes and overnight) and finally embedded in Spurr and polymerized at 60 degrees C for 16 hr. The most utilizable polar Epon mix was determined to be Epon 812 = 50 ml, NMA=42 ml, DMP-30=1-2 ml. It was somewhat brittle but cut well with both glass and diamond knives. All four polar dehydrants were found to retain lipids and carbohydrates equally well in thin section in striated and cardiac muscle, liver, kidney and brain from the rat. The E 812 was the only dehydrant that retained lung multilamellar bodies. The possible carcinogenic effects of VCD were considered and the probably metabolism and excretion of VCD were discussed.     

Flexural properties of repaired heat‐polymerising acrylic resin after wetting with monomer and acetone

doi:10.1111/j.1741‐2358.2009.00321.x
Flexural properties of repaired heat‐polymerising acrylic resin after wetting with monomer and acetone Objectives: Repair strength can be improved by treating fractured surfaces of a denture. Background: This study investigated flexural properties of heat‐polymerised acrylic resin specimens repaired with auto‐polymerising and visible light curing (VLC) resins after the repair surfaces were wetted with monomers or acetone. Materials and Methods: Fifty‐four specimens (65 × 10 × 2.5 mm) were prepared and 48 of them were sectioned to simulate denture fracture. Butt‐joint designed repair surfaces were wetted with heat‐, auto‐polymerising monomers and acetone for 180 s and repaired with auto‐polymerising and VLC resins. After repairs, specimens were subjected to three‐point bending test and flexural strength, strain, fracture load, modulus of elasticity and deflection values were recorded. Data were analysed with Student t and LSD tests (p ≤ 0.05). Results: Overall flexural strength, strain, fracture load and deflection values of specimens repaired with VLC resin were significantly higher than the specimens repaired with auto‐polymerising resin for all types of wetting agent (p < 0.05). Within the wetting agents, heat‐ and auto‐polymerising monomers produced the best mechanical properties, while wetting with acetone did not provide superior effect over both monomers. Conclusion: In clinical use, wetting the repair surfaces may result in stronger repairs. The use of bonding agent in VLC resin repairs in combination with wetting agent results in improved flexural properties.     

Immunohistologic techniques for detecting the glycolipid Gb3 in the mouse kidney and nervous system

Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membrane-bound glycolipid globotriaosylceramide (Gb(3)) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb(3) in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb(3) was affected by the method of tissue preparation. Tissue preparation included variations in fixation (immersion or perfusion) and processing (paraffin or frozen) steps; paraffin processing employed different dehydration solvents (acetone or ethanol). Perfusion-fixation in combination with frozen sections or acetone-dehydrated tissue for paraffin sections resulted in specific recognition of Gb(3) using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb(3) was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb(3) localization to endothelial cells was determined to be an artifact generated due to immersion-fixation or tissue dehydration with ethanol. This finding was corroborated by glycolipid profiles from tissue subjected to dehydration; namely Gb(3) was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and preservation of the glycolipid Gb(3) in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.     

Extracellular glycosyl hydrolase activity of the clostridia producing acetone, butanol, and ethanol

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% flour medium amounted to 12.7-15 g/l with butanol constituting 51.0-55.6%. Activities of these strains towards xylan, beta-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acertobutylicum 6, C. acetoburylicum 7, and C. acertobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824. It was demonstrated that starch in the medium could be partially substituted with plant biomass.     

A Simple Methylene Blue-Azure Ii-Basic Fuchsin Stain for Epoxy-Embedded Tissue Sections

One micron-thick sections of tissues fixed in glutaraldehyde, or in glutaraldehyde followed by osmium tetroxide, and embedded in a variety of plastic resins were stained in a methylene blue-azure II solution at 65 C, then counterstained in 0.05% basic fuchsin in 2.5% ethanol at room temperature (24 C). Considerable variation was found in methylene blue-azure II staining times for different embedding media. Aged Epon-812 required less staining time than freshly polymerized blocks of Epon-812. The procedure is a simple, rapid staining technique suitable for photomicrography and tissue orientation for electron microscopy.     

Scanning electron microscopy of heart muscle freeze-dried from dimethylsulfoxide for simultaneous demonstration of cell morphology and microvascular function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 +/- 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

Scanning Electron Microscopy of Heart Muscle Freeze-Dried from Dimethylsulfoxide for Simultaneous Demonstration of Cell Morphology and Microvascular Function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 ± 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.     

Nonvolatile chemicals provide a nest defence mechanism for stingless bees Tetragonula carbonaria (Apidae,Meliponini)

Social insects construct nests that protect their brood and food resources from both the physical environment and natural enemies. Stingless bees use plant‐derived resins, mixed with wax to form propolis, in the construction of their nests, and these products can be effective sources of defense against natural enemies, including ants. However, it is not known whether this defense, in the form of deterring or repelling workers, derives from the physical properties or chemical compounds of these products. The nest entrance of Tetragonula carbonaria is constructed with propolis and Corymbia resins, and we ask whether nonvolatile chemicals present in these products act as a defense against ants. Our field experiments revealed that workers of Iridomyrmex mayri Forel were deterred from crossing a chemical barrier comprising nonvolatile, nonpolar (hexane) extracts of propolis and Corymbia tree resin. However, polar (ethanol) extracts did not have this effect. These data are the first to demonstrate that the chemical components alone of entrance propolis and resins can provide a nest‐defense function against ants.     

Studies in lipid histochemistry

Summary A considerable portion of polar lipids survives the routine dehydration procedure for paraffin embedding with ethanol, acetone and xylene and can be detected in dehydrated blocks of tissue. Sphingomyelin, cerebrosides, sulphatides and gangliosides can be demonstrated with appropriate histochemical methods and chromatographically even in ordinary paraffin sections especially when the amount of these lipids in tissues is sufficiently high, e.g. in lipidoses and in normal myelin. In blocks of tissue dehydrated with acetone and cleared with benzen a considerably higher amount of polar lipids is present. Factors governing the preservation of polar lipids in paraffin sections are discussed.     

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mold, by Pseudomonas species on canola petals

Sclerotinia sclerotiorum is an important pathogen on canola. Due to the public concern over pesticide use, alternative methods of disease control, such as biological control, should be considered. Several bacterial strains were isolated from canola and soja plants. Inhibition of S. sclerotiorum by bacterial strains in vitro was assayed on PDA medium in dual culture test. Eight Pseudomonas sp. strains (PB-3, PB-4, PB-5, PB-6, PB-7, PB-8, PB-10 and PB-11) caused inhibition zone against 5. sclerotiorum hyphal growth. The biocontrol potential of the bacteria was tested in a plant assay. Disease suppression was investigated using a petal inoculation technique. Canola petals were pretreated with bacteria, and then inoculated with 5. sclerotiorum ascospores 24 h later. Greenhouse experiment showed that application of Pseudomonas sp. strains (1 x 10(8) cfu ml(-1)) effectively suppressed S. sclerotiorum (1 x 10(5) ascospores ml(-1)) on petals and all of them achieved significant (P<0.01) disease suppression. Fourteen days after inoculation, strain PB-3 had 88/7% disease control and strain PB-4 had 69/9% disease control. Result from all studies indicates PB-3 to be effective biocontrol against S. sclerotiorum of canola. PB-3, PB-4, PB-7, PB-8, PB-10 and PB-11 were identified as Pseudomonas fluorescens biovar III. PB-5 and PB-6 was identified as Pseudomonas fluorescens biovar II. Strains PB-3, PB-4, PB-6, PB-10 and PB-11 produced protease and HCN. Strain PB-5 produce protease; no HCN.     

Production of ethanol by adsorbed yeast cells

Summary Various ion exchange resins were tested for their ability to adsorb cells of Saccharomyces cerivisiae with the ultimate intention of developing a packed bed immobilized cell reactor for the continuous production of ethanol. The resins varied greatly in their ability to adsorb cells - the least effective resins retained less than 1 mg S. cerivisiae cells (dry weight)/g of resin (dry weight), and the most effective, 130–140 mg cells/g of resin. A column reactor packed with adsorbed yeast cells was operated continuously for over 200 hours using a 12% (w/v) glucose medium at dilution rates of 1.1 h^-1 and 1.44 h^-1 (based on void volume). High ethanol productivities of 53.1 and 62.0 g ethanol/l-h were obtained.     

Diversity matters: how bees benefit from different resin sources

Biodiverse environments provide a variety of resources that can be exploited by consumers. While many studies revealed a positive correlation between biodiversity and consumer biomass and richness, only few studies have investigated how resource diversity affects single consumers. To better understand whether a single consumer species benefits from diverse resources, we tested how the protective function of a defensive plant resource (i.e. resin exploited by social bees) varied among different sources and target organisms (predators, parasites and pathogens). To assess synergistic effects, resins from different plant genera were tested separately and in combination. We found that resin diversity is beneficial for bees, with its functional properties depending on the target organisms, type and composition of resin. Different resins showed different effects, and mixtures were more effective than some of the single resins (functional complementarity). We conclude that resins of different plant species target different organisms and act synergistically where combined. Bees that rely on resin for protection benefit more when they have access to diverse resin sources. Loss of biodiversity may in turn destabilize consumer populations due to restricted access to a variety of resources.