Plastic hood to facilitate the embedding of small embryos and tissues.
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Embryo, Mammalian↗
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Microwave oven heating enhances the ultrastructural detection of antigens in bioacryl-embedded tissue sections.
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Acrylates↗
The cell in absence of aggregation artifacts.
Eduard Kellenberger understood that the conventional resin-embedding, he helped to develop (Ryter, A., Kellenberger, E., 1958. L'inclusion au polyester pour l'ultramicrotomie. J. Ultrastruct. Res. , 2, 200-214), was prone to aggregation artifacts (Kellenberger, E., 1987. The response to biological macromolecules and supramolecular structures to the physics of specimen cryo-preparation. In: Steinbrecht, R.A., Zierold, K. (Eds.), Cryo-techniques in Biological Electron Microscopy, Springer, Berlin, pp. 35-63). He was instrumental in developing various methods to overcome this limitation, for instance, by using low temperature-embedding and partially hydrophilic resins (Carlemalm, E., Garavito, R.M., Villiger, W., 1982. Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microstruct., 126, 123-143; Villiger,W., 1993. Low temperature-embedding with Lowicryl resins. In: Robards, A.W., Wilson, A.J. (Eds.), Procedures in electron microscopy, Wiley, Chichester, UK, pp. 16:7.3-16:7.6). In principle, cryo-electron microscopy of vitreous sections is free of any aggregation artifact since the material remains fully hydrated and is free of chemical fixation or staining. The method is technically difficult still, but recent progress has made it amenable to routine practical applications. We compare here electron microscopical aspects of Zea mays meristem cells prepared by: (1) conventional resin-embedding and sectioning; (2) low temperature-embedding and sectioning of freeze substituted samples; and (3) cryo-sections of vitrified samples. The appearance of the extra-cellular space, the cytoplasm and the nucleoplasm are very different in conditions (1) and (3). They appear as compact, irregular and well delineated structures in conventional resin sections, whereas they are more diffuse and homogeneous in the vitreous sections. In the resin sections, the material seems to form a complex matrix, whereas it looks more like a thick soup in the vitreous sample. Low temperature-embedding (condition 2) shows an intermediate appearance. We suggest that regardless of the difference due to staining and different sectioning conditions, the other image differences are the consequence of aggregation artifacts in the resin-embedded specimens.
Artifacts↗
Cold acetone fixation and methacrylate embedding. A suitable method for routine processing of bone marrow biopsies.
Here we report that acetone fixation at -18 degrees C with subsequent embedding in methyl-/butylmethacrylate is a reliable method for the routine processing of bone marrow biopsies. This method allows good conventional histological visualization of morphological details, which is comparable with other fixation procedures. The essential advantage of this method is that a wide range of monoclonal antibodies and polyclonal antisera can be used for immunohistochemical investigations for diagnostic and scientific purposes. The addition of 5% polyethylene glycol 400 to the acetone minimizes freeze-related artefacts. The immunohistochemical demonstration of a number of antigens is mostly affected by the medium used for slide preparation and to a lesser extent by the concentration of benzoylperoxide used for polymerization. Performing polymerization at 4 degrees C and using N, N-dimethyl-p-toluidine as accelerator allows the concentration of benzoylperoxide to be reduced to 0.2 g% (8.3 mmol). Under these conditions the methacrylate embedding procedure has only minimal effects on the quality of immuno- and enzyme histochemistry. Additionally, the simplified method for removing the polymerization inhibitor from the methacrylate components and the shortened impregnation step are further advantages of the embedding method described here.
Acetone↗
A simplified method for obtaining 0.5-microns sections of small tissue specimens embedded in PEG.
We describe a new method for embedding small specimens in polyethylene glycol (PEG) 4000. This method preserves cell morphology and provides sensitive immunocytochemical labeling with excellent subcellular resolution. Small tissues are embedded in agarose so that they can be grouped together and oriented for sectioning before infiltration with PEG 4000, a water-soluble polymer. Fixation, embedding, sectioning, and staining can be performed in 1 day. Results from immunocytochemical studies localizing actin and tubulin on 0.5-micron sections of PEG-embedded specimens are compared with those obtained on semi-thin sections of araldite-embedded specimens and demonstrate the ease, speed, and increased sensitivity of this embedding method.
Actins↗
A method for the study of undecalcified bones and teeth with attached soft tissues. The Säge-Schliff (sawing and grinding) technique.
A new sawing-grinding method is described for the histological evaluation of jaw bones with teeth or bones containing implants (ceramic or metallic). The undecalcified bone is embedded in acrylic resin and sawed at 100 to 150 micrometers. The slices are ground automatically by a special machine to a thickness of 5-10 micrometers. The usually employed staining procedures for hard plastic embedded-tissues may be used. Plaque, fillings, crowns, bridges, implants and soft tissues are preserved in situ. Macroscopic and microscopic detail of good quality is preserved for histological and morphometrical evaluation.
Adult↗
Polychromatic staining of epoxy semithin sections: a new and simple method.
A simple, rapid method is described for the polychromatic coloration of semithin sections, which is applicable to material routinely processed for transmission electron microscopy. Material fixed with a glutaraldehyde-paraformaldehyde mixture and postfixed in osmium tetroxide with or without potassium ferrocyanide and embedded in different types of resin (Durkupan-ACM, Spurr resin, Taab resin) can be used. Constant and homogenous results are obtained with this technique, the staining procedure being achieved at room temperature in no more than 10 min. Sections of 0.5-1 microns in thickness are oxidised and bleached. After washing, sections are stained in two steps with carbol methylene blue/carbol gentian violet solution and pararosaniline solution. Using the method described in this paper, a polychromatic coloration of the different cells and tissues was obtained (epithelial cells in various shades of blue-violet, connective tissue and elastic laminae of blood vessels in pink or red, etc.). This procedure provides greater contrast between cytoplasm and nuclei, and among the different types of cells and tissues than is seen with toluidine blue, which is very useful for observation and photography of semithin sections. Polychromatic methods found in the literature are normally complex and require a lengthy staining time or cannot be applied on material routinely processed for transmission electron microscopy. Our method is simple, rapid and can be used on any type of material routinely processed for transmission electron microscopy and embedded in epoxy resins.
Animals↗
The embedding of ossicular chain specimens in plastic.
The following objects were specifically designed for teaching material to the medical students. Dried human auditory ossicles were bleached in 30% hydrogen peroxide solution and then, embedded in a good quality plastic. The plastic mass was shaped into a ball 5 cm in diameter, and the surface of which was polished with a good finish. The merits of the objects may be listed as follows; 1) A permenant preservation of the objects is possible because they are embedded in heat- and chemical-resistant material. 2) The objects are handy for use in the lecture room and easily observed by the students because of the magnifying effect of the plastic covering.
Anatomy↗
Clearing and embedding in polyester resin for demonstrating the nerve distribution pattern of skeletal muscles.
The preservation of many stained gross specimens in solution creates some difficulties. It is convenient and effective to preserve material in polyester resin instead of glycerol. The aim of this study was to determine the usefulness of clearing and embedding using polyester resin. The samples consisted of the nerve distribution patterns of skeletal muscles stained using Sihler's method. The muscles were cleared more successfully and the intramuscular nerve distributions were demonstrated better in polyester than in glycerol. The method presented here eliminates not only the storage and handling problems of specimens, but also problems such as pale stains and the molding of preparations. Furthermore, it is more convenient to examine and to photograph specimens cleared and embedded in polyester than those stored in glycerol.
Glycerol↗
Glycol methacrylate embedding of bone marrow cores.
The objective of the present study was to assess the use of glycol methacrylate (GMA) as an embedding medium for bone marrow biopsies. Paraffin-embedded bone marrow trephines, which are most widely used for diagnostic purposes, need to be decalcified prior to sectioning. This process has a number of effects, and results in interference with the evaluation of the bone marrow. Differentiation of some cases of hematological malignancies may be difficult in decalcified, paraffin-embedded bone marrow biopsy specimens, because of the reported inability to obtain reproducible blast counts, and because a considerable percentage of cells cannot be easily recognized. On the other hand, GMA-embedding of marrow cores, which obviates the need for decalcification, shows structural organization of the marrow and gives excellent cellular details. The simplicity of GMA embedding, short processing time and reproducibility, makes this method highly suitable for routine work in the hematology laboratory.
Biopsy↗
A neovascularized organoid derived from retrovirally engineered bone marrow stroma leads to prolonged in vivo systemic delivery of erythropoietin in nonmyeloablated, immunocompetent mice.
Marrow stromal cells (MSCs) are postnatal progenitor cells that can be easily cultured ex vivo to large amounts. This feature is attractive for cell therapy applications where genetically engineered MSCs could serve as an autologous cellular vehicle for the delivery of therapeutic proteins. The usefulness of MSCs in transgenic cell therapy will rely upon their potential to engraft in nonmyeloablated, immunocompetent recipients. Further, the ability to deliver MSCs subcutaneously - as opposed to intravenous or intraperitoneal infusions - would enhance safety by providing an easily accessible, and retrievable, artificial subcutaneous implant in a clinical setting. To test this hypothesis, MSCs were retrovirally engineered to secrete mouse erythropoietin (Epo) and their effect was ascertained in nonmyeloablated syngeneic mice. Epo-secreting MSCs when administered as 'free' cells by subcutaneous or intraperitoneal injection, at the same cell dose, led to a significant - yet temporary - hematocrit increase to over 70% for 55+/-13 days. In contrast, in mice implanted subcutaneously with Matrigel trade mark -embedded MSCs, the hematocrit persisted at levels >80% for over 110 days in four of six mice (P<0.05 logrank). Moreover, Epo-secreting MSCs mixed in Matrigel elicited and directly participated in blood vessel formation de novo reflecting their mesenchymal plasticity. MSCs embedded in human-compatible bovine collagen matrix also led to a hematocrit >70% for 75+/-8.9 days. In conclusion, matrix-embedded MSCs will spontaneously form a neovascularized organoid that supports the release of a soluble plasma protein directly into the bloodstream for a sustained pharmacological effect in nonmyeloablated recipients.
Animals↗
Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver.
Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5'-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), D-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 microns in thickness. For all the enzymes that could be detected, the 6 microns:3 microns ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.
Animals↗
Blister artifact formation due to organic solvent effects on Spurr's epoxy resin semithin sections.
Wrinkles and air bubble artifacts may occur when preparing slides of semithin sections (0.5 microm) from blocks embedded in different resins. More than aesthetically annoying, wrinkles and air bubble artifacts may prohibit study of small structures. Present observations suggest that organic solvent based mounting media may interact with the resin of the section. This sometimes causes wrinkles and air bubble artifacts in the sections that degrade the quality of light microscope images. We compared the quality of semithin sections of several tissues in different resins using various types of mounting media. We observed that sections from Spurr's resin have many more artifacts. In particular, small, 2-10 microm round or oblong blister-like artifacts often plague our Spurr's resin sections. We demonstrate that Spurr's resin sections react with toluene and xylene in organic solvent based mounting media forming blisters, while sections from Araldite and L. R. White do not. We suggest combinations of embedding and mounting media for successful preparation of semithin sections for light microscopy without wrinkles, blisters, or air bubble artifacts.
Animals↗
Application of cryosubstitution in neurohormone- and neurotransmitter-immunocytochemistry.
Pituitaries of the African catfish, Clarias gariepinus, were prefixed in aldehyde fixatives, frozen in liquid propane and submitted to a cryosubstitution procedure. Ultrathin sections of the Lowicryl HM20-embedded tissue were treated with primary antisera raised in rabbits to gonadotropin releasing hormone (GnRH), vasopressin or gamma amino butyric acid (GABA) respectively. Binding of the primary antisera was visualized with goat anti-rabbit (GAR) labeled with gold. The general morphology of the tissue components in the cryosubstituted pituitaries matches with that obtained after routine embedding procedures. In addition, a strong labeling intensity of the neuropeptides/neurotransmitters investigated in the present study was demonstrated. Due to these qualities cryosubstitution provides optimal conditions for studying co-localization of neurosecretory products, using double-immunostaining procedures. In the pars distalis of the catfish pituitary several types of hypothalamus-derived nerve fibers are present between or synapting on the secretory cells. It is demonstrated that the two known catfish GnRHs are co-localized in the same nerve fiber and within these nerve fibers even co-exist in the same neurosecretory granules. GABA and vasopressin-immunolabeling each occurred in different nerve fibers. The present data demonstrate that cryosubstitution and low temperature-embedding results in an excellent morphological preservation compared to ultracryotomy and a better preserved immunoreactivity of small antigenic molecules in comparison to conventional fixation and embedding techniques.
Animals↗
A method for processing fluorescent labelled tissue into methacrylate: a qualitative comparison of four tracers.
A technique for preserving fluorescence in retrogradely labelled neurons embedded in resin was developed. Four retrograde tracers were tested, Fast Blue (FB); Diamidino Yellow (DY); tetramethylrhodamine dextran (fluoro-ruby) (TMRD) and fluorescein dextran (fluoro-emerald) (FD). These tracers were applied to the cut end of the sciatic nerves in rats either by: (a) direct application of tracer crystals, or (b) dipping the nerve into an aqueous solution containing the tracer. Each lumbar spinal cord was removed and dehydrated by one of two methods: (a) conventional alcohol dehydration, or (b) dehydration through a graded series of aqueous methacrylate infiltration solutions (inert dehydration). Specimens were embedded in methacrylate and horizontal sections cut. The location of labelled motoneurons was mapped using a fluorescence microscope. Direct application of tracer crystals labelled more motoneurons than dipping. Fast Blue labelled considerably more motoneurons than tetramethylrhodamine. Labelling by all tracers was retained following methacrylate embedding. Fast Blue and Diamidino Yellow required inert dehydration, while tetramethylrhodamine dextran and fluorescein dextran were preserved using conventional dehydration. These results indicate that tissue labelled with commonly used fluorescent tracers can be processed and embedded in methacrylate, thereby permitting quantitative analysis by modern stereological methods.
Animals↗
A simple wax-embedding method for isolation of aphid hemolymph for detection of luteoviruses in the hemocoel.
A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid. Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus (BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively. A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and their insect vectors.
Animals↗
A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno-electron microscopical studies.
A combination of cryofixation without pre-treatment, freeze-substitution and low-temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P.chrysogenum colonies were grown between dissected-dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze-substitution media, but it was reduced after embedding in Epon 812.
Acyltransferases↗
A crown reconstructing method for resin-embedded transparent teeth.
OBJECTIVE: Resin-embedded transparent teeth are devoid of the original crown morphology, because enamel is lost by demineralization. This study was designed to reproduce artificial enamel and to reconstruct the original crown morphology for resin-embedded transparent teeth. METHOD AND MATERIALS: The impression of the coronal portion of human permanent teeth was taken with a silicone impression material. After demineralization, drawing ink was injected into the pulp cavities. The ink-infiltrated teeth were made transparent with methyl salicylate and embedded with polyester resin. Urethane prepolymer was injected into the impression, and the resin-embedded teeth were reinserted into the impression. After polymerization of the urethane resin, the specimens and the urethane resin were removed from the impression. RESULTS: The original crown morphology of the resin-embedded transparent teeth could be precisely reconstructed with artificial and removable enamel. The resin-embedded teeth showed morphologic details of the black-stained pulp cavities through the transparent dentin and cementum. CONCLUSION: This study established a crown reconstructing method for resin-embedded transparent teeth. The specimens will be useful for demonstration of morphology of teeth and pulp cavities.
Carbon↗