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At least 937 records · Page 52Linked to original sources
PubMed1999

Resins for combined light and electron microscopy: a half century of development.

The last fifty years have seen enormous improvements in the way biological specimens are prepared for microscopy. The Fifties produced the essential groundwork upon which many of our current methodologies are based. Acrylic resin embedding was introduced in 1949, with subsequent publications seeking improvements to resin formulations, embedding protocols, and modes of polymerisation. Procedures for progressive lowering of temperature processing, cryosubstitution, freeze-drying and polymerisation by ultra-violet light at low temperatures, all had their genesis in this decade of great innovation. The Sixties marked the period when the acrylics were eclipsed by the more stable and reliable epoxy resins, and much of our present-day understanding of ultrastructure was elucidated. The Seventies carried on this work with advances in technical developments concerned mainly with freezing methodologies. The beginning of the Eighties saw a resurrection of the acrylic resins, with new formulations of these resins giving reliable and stable embeddings. The low temperature and freezing methodologies pioneered in the Fifties, backed up by recent improvements to low temperature technologies, were used to further our understanding of ultrastructure and breathe new life into the science of immunocytochemistry. The remainder of the Eighties and Nineties has seen the ever increasing application of these various microscopical techniques to a wide range of biological studies. The flexibility offered by the acrylic resins in choosing between different processing, embedding and polymerisation methods has provided the impetus for detailed studies to bring to the attention of microscopists the underlying trends governing specimen preparation. Therefore, looking forward to the new Millennium, this has allowed for a more reasoned choice in organising a strategy to deal with a variety of microscopical requirements and for planning an appropriate protocol.

Catalysis↗
PubMed1993

Cryofixation and cryosubstitution: a useful alternative in the analyses of cellular fine structure.

A study of the fine structure of cultured mouse P815 cells as well as of mouse liver tissue after having undergone cryofixation and cryosubstitution is reported here. The P815 cells were cryofixed by a projection onto a liquid nitrogen (LN2), or liquid helium (LHe), cooled copper mirror: the liver tissue was processed (cryofixed) by high pressure freezing using a Balzers HPM 010 apparatus. No conventional chemical fixatives were used in the substitution medium which consisted of pure acetone. Embedding was carried out either in Lowicryl K11M, Lowicryl HM23, Epon or in LR White resins. The results of this study enabled us to conclude that a) one can obtain good preservation of cellular ultrastructure using different cryofixation methods and cryosubstitution without the use of chemical fixatives: b) different methods of embedding can be applied after various cryofixation techniques giving rise to slight differences in the well preserved fine structure; and c) high pressure freezing is to be recommended, in cryosubstitution studies, over that of slam freezing especially when cryofixing larger pieces of tissue which can result in a good morphology of up to 400 microns in depth.

Animals↗
PubMed1993

New staining methods for semithin sections of tissues of Seriola dumerili embedded in methacrylate.

Semithin sections of different tissues of Seriola dumerili R. Embedded in glycol methacrylate, were stained with two staining methods normally used in conventional haematology and histology: Gutierrez's cytopanchrome and Gutierrez's polychrome V.O.F. The results observed in all tissues embedded in this resin and stained with V.O.F. are satisfactory and comparable, as to staining affinity, to those obtained on paraffin-embedded tissues. Gutierrez's trichrome stain appears to be a useful tool in the study of oogenesis for detecting changes in staining according to cellular structure and maturation stage. The results observed with cytopanchrome are similar to those obtained with toluidine blue in glycol methacrylate-embedded tissues. The use of these two staining techniques, requires no previous oxidation, alkali treatments, high temperatures or removal of the embedding medium. Furthermore, both dyes can be used at the same time: in this case, cytoplasm of previtellogenic oocytes showed a stronger affinity for cytopanchrome, and were more intensely stained by V.O.F. as the vitellogenesis of Seriola dumerili progressed. Both stain, and especially the cytopanchrome one, also are available for staining the liver, gills and testis.

Animals↗
PubMed1995

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate in Epon embedded specimens.

A comparison between the time-consuming freeze-substitution Lowicryl HM 20 embedding procedure with the Epon embedding method for the immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate (PIP2) by means of a monoclonal antibody is described. The results indicate that the intracellular localization of the phospholipid and the efficiency of its identification are similar, opening the possibility of performing the immunoreaction also on samples that are routinely processed for pathological investigations.

Animals↗
PubMed1995

Immunohistochemical detection of epidermal growth factor in glycol methacrylate embedded tissue.

This study addresses a variety of immunohistochemical conditions for detecting EGF in 3.5% paraformaldehyde fixed, glycol methacrylate embedded tissue including antigen unmasking with trypsin, dilution of primary antibody, and incubation time with primary antibody. Color development was achieved with a biotinylated secondary antibody linked to an avidin biotinylated peroxidase complex. Trypsinization and a 12 hr incubation with the primary antibody was essential to detect EGF in this system. Adequate staining could be achieved with a 1:100 dilution of the primary antibody.

Animals↗
PubMed1997

Sheet plastination of the vascularity of the lunate bone--a morphological study.

Using the method of sheet plastination, the vascularity of the lunate bone can be investigated and analyzed. By intraosseous injection, the deep and superficial venous systems are visualized. Transparent sheets with slices of embedded specimens show a three-dimensional presentation of the vascularity of the human carpus different in functional aspects. Comparing hands with a normal and extended wrist position, three mechanisms of impairment of the venous drainage are observed. The anatomical results of this investigation support the hypothesis that venous congestion, resulting from intraosseous hypertension, plays a role in the development of the necrosis of the lunate bone.

Drainage↗
PubMed1999

Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and ultrastructural (EM) study.

We developed an improved method for the detection of double-strand DNA breaks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebellar granule cells were exposed to low potassium conditions to induce apoptosis. Twenty-four hr after treatment, one group of cells was fixed in situ with 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish, pelleted, fixed with a 4% paraformaldehyde and 0. 2% glutaraldehyde mixture, and embedded in LR White. For LM, the modified TUNEL technique was performed on 1.5-microm LR White sections and apoptotic cells were visualized using an enzymatic reaction to generate a blue precipitate. For EM, thin sections (94 nm) were processed and DNA fragmentation was identified using modified TUNEL with streptavidin-conjugated gold in conjunction with in-depth ultrastructural detail. Alternate sections of cells embedded in LR White can therefore be used for LM and EM TUNEL-based detection of apoptosis. The present findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fundamental mechanism of cell death. (J Histochem Cytochem 47:561-568, 1999)

Animals↗
PubMed2000

Embedding thin plant specimens for oriented sectioning.

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.

Acrylic Resins↗
PubMedDate not supplied

Correlative light and backscattered electron microscopy of bone--Part I: Specimen preparation methods.

A method for preparing nondecalcified bone and tooth specimens for imaging by both light microscopy (LM) and backscattered electron microscopy in the scanning electron microscope (BSE-SEM) is presented. Bone blocks are embedded in a polymethylmethacrylate (PMMA) mixture and mounted on glass slides using components of a light-cured dental adhesive system. This method of slide preparation allows correlative studies to be carried out between different microscopy modes, using the same histologic section. It also represents a large time savings relative to other mounting methods whose media require long cure times.

Adhesives↗
PubMed1994

Demonstration of Sindbis virus antigen in Lowicryl-embedded cultured cells by immunogold staining.

Eight monoclonal antibodies (MAbs) were tested for the use in immunogold staining of Sindbis virus (SIN) antigen in cultured Baby Hamster kidney (BHK) cells. The antibodies were directed against the capsid (C) protein (5) or against the glycoprotein E1 (3), respectively. Four out of five anti-C MAbs and two out of three anti-E1 Mabs reacted on frozen sections, whereas two of the anti-C antibodies and none of the anti-E1 antibodies reacted on Lowicryl K4M-embedded cells. The MAbs reacting, in particular the anti-C Mabs which react on Lowicryl K4M-sections, will allow further studies of the pathogenesis and replication of alphaviruses on the cellular level. The results also confirm the conformation-dependence of the glycoprotein epitopes.

Acrylic Resins↗
PubMed2006

Site-specific 3D imaging of cells and tissues with a dual beam microscope.

Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.

Animals↗
PubMed2003

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum.

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.

Anatomy, Cross-Sectional↗
PubMed2001

Effect of total body irradiation on peri-implant tissue reaction: an experimental study.

The aim of the present study was to evaluate the osseointegration process under total body irradiation conditions (LD 50/30). Twenty Wistar rats (mean body weight: 90 g) were used. Under ethyl urethane intraperitoneal anesthesia (1 g/kg body weight), the animals were irradiated with a single 700 cGy dose (linear accelerator 6 Mev photons). Four days post irradiation, a titanium laminar implant was placed in the left tibia of each rat. Antibiotic therapy (ceftriaxone) was administered daily post implantation, to prevent infection by radiation. Fourteen days post implantation, the animals were killed by ether overdose. The tibiae were resected, radiographed and processed for embedding in methyl methacrylate. The results showed impaired osteogenesis and absence of osseointegration in experimental tibiae. This could be due to a direct action of total body irradiation on osteogenesis precursor cells. This effect would impair bone formation involved in peri-implant osseointegration processes in this experimental model.

Animals↗
PubMed2002

Freeze-dried and resin-embedded biological material is well suited for ultrastructure research.

Transmission electron micrographs of different biological material, cryofixed, freeze-dried and embedded in Spurr's resin, in Epon, or in Lowicryl, are presented. The structure preservation obtained either without or with application of chemical fixatives after drying showed that freeze-dried embedded specimens are particularly well suited for new morphological, immunocytochemical and microanalytical studies aimed at detecting the life-like subcellular distribution of mobile macromolecules and ions. The results also indicate that the removal of cell water by freeze-drying from the areas of best cryofixation is relatively slow. Ultrathin sections of well cryofixed biological material embedded after freeze-drying in Spurr's resin or Epon reveal cellular plasma phases with very fine granularities and well defined membranes in negative contrast. This may be due to the preservation of the original structure of cellular macromolecules with a considerable amount of their hydration water. Sublimation studies with differently hydrated and cryofixed macromolecules are suggested to settle this issue.

Acrylic Resins↗
PubMed1992

Electron spectroscopic study (ESI, EELS) of Nanoplast-embedded mammalian lung.

The potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). Samples were either fixed with glutaraldehyde-paraformaldehyde or glutaraldehyde-tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted. A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast-embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L2,3 edge observed by EELS of Nanoplast-embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast-embedded samples by computer-assisted analysis of electron spectroscopic images. In this respect glutaraldehyde-paraformaldehyde fixation is preferable to glutaraldehyde-tannic acid fixation because the presence of silicon, revealed by EELS, in tannic-acid-fixed samples may introduce artefacts in phosphorus distribution images obtained by the three-window method because of the close proximity of the L2,3 edges of silicon and phosphorus.

Animals↗
PubMed1997

Use of methyl methacrylate resin for embedding bone marrow trephine biopsy specimens.

AIMS: To evaluate the use of methyl methacrylate resin as an embedding medium for undecalcified bone marrow trephine biopsy specimens. METHODS: About 2500 undecalcified bone marrow trephine biopsy specimens were processed, and embedded in methyl methacrylate resin. Semithin sections (2-3 microns) were stained by routine tinctorial and immunocytochemical staining methods with a wide range of antibodies using a standard streptavidin biotin horseradish peroxidase technique. Different antigen retrieval pretreatments were evaluated. RESULTS: Bone marrow trephine biopsy specimens are embedded routinely in methyl methacrylate at the Haematological Malignancy Diagnostic Service at The Leeds General Infirmary. Over 50 different primary antibodies are in current use; for the majority of these, microwave antigen retrieval or trypsin digestion, or both, is either essential or greatly enhances the results. CONCLUSIONS: Embedding bone marrow trephine biopsy specimens in methyl methacrylate resin retains morphology and permits reliable, high quality immunocytochemistry. This is particularly desirable for the demonstration of neoplastic cells in regenerative marrow after chemotherapy, and in the detection of residual disease after treatment. The use of methyl methacrylate for routine use on bone marrow trephine biopsy specimens is advocated.

Biopsy↗
PubMed1996

Distribution of concanavalin A and wheat germ agglutinin binding sites in the rat peripheral nerve fibres revealed by lectin/glycoprotein-gold histochemistry.

The affinity of rat peripheral nerve fibres for concanavalin A (Con A) and wheat germ agglutinin (WGA) was tested in semi-thin sections of Epon-embedded material. A two-step post-embedding technique was used. As a first step, Con A and WGA were used in pure form. As a second step, peroxidase-gold (for Con A) and ovomucoid-gold (for WGA) complexes were applied. The lectin-binding sites, visualized by means of signal amplification with the photochemical silver reaction, were associated mainly with the myelin sheaths and the surfaces of Schwann cells.

Animals↗
PubMed2000

Minimal compression of ultrathin sections with use of an oscillating diamond knife.

With the aim to minimize compression artefacts in ultrathin sections, coincident with the stroke direction, we have invented an oscillating diamond knife. Results and theoretical considerations explaining its function are discussed. During conventional ultrathin sectioning the resultant compression is in the order of 20-35% of section height. This holds true for sections of samples embedded into Lowicryl HM20 and of the polymer polystyrene, cut with a 45 degrees diamond knife and floated on water. The oscillating knife reduces this compression almost completely. It consists of a diamond knife on which a low voltage piezoelectric translator (piezo) is mounted, which oscillates when the piezo is driven by an alternating voltage source. No additional cutting artefacts were observed in the micrographs when they were compared with sections produced without oscillating the knife.

Acrylic Resins↗