Plastic Embedding Research Articles

Bone histomorphometry in three females with Rett syndrome

The frequent occurrence of osteoporosis in Rett syndrome raises questions about the etiology of this finding. It is unknown whether there is any relationship between low bone mass and the underlying genetic disorder. We recently had an opportunity to study the status of bone remodeling by quantitative bone histomorphometry in three girls ages 9.75, 13.5, and 14 years, with typical Rett syndrome who required scoliosis surgery. Anterior iliac crest bone biopsies were performed 1–2 days after double labeling of the bone surfaces with tetracycline. Samples were processed for plastic embedding, sectioned, stained, and histomorphometry performed in the cancellous bone. The same observer performed all measurements. Bone volume was reduced, surface parameters of formation (osteoid surface) were normal while parameters of resorption (osteoclast surface and number) were decreased. The rate of bone formation was reduced in the first two girls but could not be measured in the third girl due to lack of double labeling. It may be that the slow rate of bone formation seen in each age group impedes the development and accumulation of peak bone mass and contributes to the decreased bone volume associated with Rett syndrome, although the data is limited. This is the first report to document decreased bone volume determined by quantitative bone histomorphometry in patients with Rett syndrome. With the recent identification of MECP2 mutations in Rett syndrome it is quite likely that genetic factors not only play a major role in brain development but may also influence other organ growth including bone formation.

Force Modulation Elasticity Mapping of Plastic-embedded, Thin-sectioned Skeletal Muscle

AbstractWe have been researching the capability of atomic force microscopy to reveal nontopographic properties of tissue embedded in plastic and sectioned with standard electron microscopic techniques. We present topography and elasticity maps of plastic-embedded, thin sections of muscle tissue. The images show topography correlated with the normal repeating structure of the sarcomere. Elasticity mapping using force modulation revealed contrast between the actin- and myosin-rich areas. We attribute the observed contrast in elasticity to the difference in local concentrations of biological material in embedding plastic.

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens

In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White® resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White® embedded tissues to be sectioned at 10 μm providing excellent signal contrast.

22] Subcellular localization of Rab17 by cryo-immunogold electron microscopy in epithelial cells grown on polycarbonate filters

This chapter discusses the subcellular localization of Rab17 by cryo-immunogold electron microscopy in epithelial cells grown on polycarbonate filters. High-resolution cryo-immunogold EM is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Fixed material is embedded in gelatin, cryosectioned, mounted on Formvar-coated grids, and labeled. A typical labeling protocol is described based on the work by Slot and Geuze during which the sectioned material is exposed first to antibody, then to protein A-gold particles. If the primary antibody does not bind to protein A, incubation with a secondary antibody that does bind protein A can be added. The precise reaction conditions used for immunogold labeling may vary depending on the antibody and are determined by trial and error.

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens

TEMPORAL DEVELOPMENT OF 2′,3′‐DIDEOXYINOSINE (DDI)‐INDUCED PERIPHERAL MYELINOPATHY

The anti‐HIV therapeutic dideoxyinosine (ddI) has been reported to produce a painful, dose‐limiting peripheral myelinopathy in HIV‐infected patients after chronic administration. We have previously demonstrated ddI‐induced myelinopathy in a non‐HIV‐infected rat model after 20 weeks of dosing, characterized by myelin splitting and intramyelin edema. The present study examined the time course needed to produce the ddI‐induced neuropathy. Adult male Sprague‐Dawley rats were gavaged with vehicle of 415 mg/kg ddI twice daily for up to 20 weeks. Groups of treated (n = 6‐8) and control (n = 3–5) animals were killed after 5, 10, 15, and 20 weeks of dosing and the distal end of the sciatic nerve was removed. The nerve was postfixed by immersion in neutral phosphate‐buffered formalin, dehydrated in graded alcohols, and embedded in plastic embedding media. One‐micrometer‐thick sections were cut and stained with toluidine blue and basic fuchsin. Plasma levels of ddI on the day the animals were killed were greater than 10 mu g/ml within the first hour after dosing and fell rapidly to less than 1 mu g/ml (clinical range 1–2 mu g/ml) within 3 h after dosing. The abnormalities observed in the sciatic nerve were few, if any, after 5 or 10 weeks, but very prominent after 15 weeks of dosing. Four of the six ddI‐treated rats exhibited abnormal morphology as evidenced by myelin splitting and ballooned myelin sheaths. Although abnormal morphology was present at 20 weeks of dosing, the effect was not as robust as at 15 weeks. This suggests that the nerve may partially recover from the effects of ddI with time.

Applications of plastic embedding to electron microscopic immunocytochemistry and in situ hybridization in observations of production and secretion of peptide hormones.

Plastic embedding has been used to localize various antigens in conjunction with immunohistochemistry. Peptide hormones have been among the antigens that have been studied extensively. Recent application of water-soluble plastics such as LR White and Lowicryl has extended the ranges of detectable antigens and enabled the observation of antigen-antigen or mRNA-antigen combinations. This review article deals with technical aspects, procedures, and applications in endocrine cells.

Temporal development of 2′,3′-dideoxyinosine (ddI)–induced peripheral myelinopathy

The anti-HIV therapeutic dideoxyinosine (ddI) has been reported to produce a painful, dose-limiting peripheral myelinopathy in HIV-infected patients after chronic administration. We have previously demonstrated ddI-induced myelinopathy in a non-HIV–infected rat model after 20 weeks of dosing, characterized by myelin splitting and intramyelin edema. The present study examined the time course needed to produce the ddI-induced neuropathy. Adult male Sprague–Dawley rats were gavaged with vehicle or 415 mg/kg ddI twice daily for up to 20 weeks. Groups of treated ( n = 6–8) and control ( n = 3–5) animals were killed after 5, 10, 15, and 20 weeks of dosing and the distal end of the sciatic nerve was removed. The nerve was postfixed by immersion in neutral phosphate-buffered formalin, dehydrated in graded alcohols, and embedded in plastic embedding media. One-micrometer-thick sections were cut and stained with toluidine blue and basic fuchsin. Plasma levels of ddI on the day the animals were killed were greater than 10 μg/ml within the first hour after dosing and fell rapidly to less than 1 μg/ml (clinical range 1–2 μg/ml) within 3 h after dosing. The abnormalities observed in the sciatic nerve were few, if any, after 5 or 10 weeks, but very prominent after 15 weeks of dosing. Four of the six ddI-treated rats exhibited abnormal morphology as evidenced by myelin splitting and ballooned myelin sheaths. Although abnormal morphology was present at 20 weeks of dosing, the effect was not as robust as at 15 weeks. This suggests that the nerve may partially recover from the effects of ddI with time. Published by Elsevier Science Inc.

Application of plastic embedding for sectioning whole-mount immunostained early vertebrate embryos.

Development of tissues and organs relies on the constant interplay of intracellular events and molecules in the microenvironment of the cells. Dynamics of patterning of molecules in a spatiotemporal manner become important in understanding any developing system. Immunohistochemistry has become an important technique for the study of specific proteins in the early embryo. Ability to localize molecules in the embryo with the greatest resolution possible provides much critical information regarding intracellular and extracellular localization of specific molecules and changes in their patterning over time. For example, an apical or basal cellular localization of a molecule may be critical in relation to its function. Thus, the definition and correlation of the spatiotemporal patterns of protein expression in the developing embryo with events of morphogenesis may give useful information on the possible roles of these molecules. Such analyses will provide a more precise basis for further experimentation. To obtain the highest resolution possible in antigen localization, a pre-embedding immunohistochemical technique followed by whole-mount plastic embedding and sectioning at 1 μm has been modified to make it useful for analyzing events of early chick and mouse morphogenesis in specific regions of the embryo. The description of the pre-embedding immunohistochemistry method and embedding procedure provided is an adaptation of the technique of Franklin and Martin (1) and others (2,3). This technique has been used with good results for analyses of whole chick embryos up to 48 h of development and for whole mouse embryos up to 9.5 d of gestation and isolated organs (i.e., heart) up to day 15 of gestation. For presentation purpose, this chapter is divided into two parts, dealing with chick embryos and mouse embryos, respectively.

Inflammatory aetiology of primary oesophageal achalasia: an immunohistochemical and ultrastructural study of Auerbach's plexus.

Achalasia is a disease of the oesophagus characterized by increased lower oesophageal sphincter (LOS) tone, absence of LOS relaxation with swallowing and aperistalsis of the body of the oesophagus. The aetiology and pathogenesis of idiopathic achalasia is still controversial. We examined 16 oesophageal biopsies and one low oesophagectomy specimen from patients with achalasia. The control group was composed of five autopsy cases with no history of oesophageal disorders, three cases of diffuse oesophageal spasm, one of gastro-oesophageal reflux disease and one patient with oesophageal carcinoma. Sections were immunostained for neurofilaments NF70 and NF200, S100 protein and neurone-specific enolase. Biopsies with inflammatory infiltrates, were in addition immunostained with antibodies against leucocyte common antigen as well as for CD20, CD43, CD68 and CD45RO. All biopsies were examined after plastic embedding, and electron microscopy (EM) was performed on samples containing autonomic plexus. An inflammatory infiltrate of varying intensity was present along the nerve fascicles and around ganglion cells in 90% of the cases of achalasia. T-lymphocytes predominated in all these cases. The autonomic nerves showed loss of fibres and degenerative changes which were discernible only by EM. Although there was no convincing neuronal loss or signs of active neuronal degeneration in biopsied cases, the oesophagectomy specimen revealed total absence of neurones and significant loss of nerve fibres. The control group showed normal plexuses and no inflammation. Degeneration and significant loss of nerve fibres associated with predominant T-cell lymphocytic inflammatory infiltrate around the myenteric plexus support the concept for the inflammatory, probably autoimmune, aetiology of autonomic nervous system injury in primary achalasia.

A study of the gonadotropin releasing hormone neuronal network in the median eminence of the rhesus monkey ( Macaca mulatta) using a post-embedding immunolabelling procedure.

The purpose of this study was to describe the ultrastructural features of gonadotropin releasing hormone (GnRH) axonal processes in the median eminence of the monkey, using a post-embedding immunogold labelling procedure. Evidence was also sought to evaluate the view that release of this peptide may be governed by direct inputs to GnRH axons in the median eminence. Plastic embedding was used to preserve ultrastructure, and a polyclonal rabbit anti-GnRH was used as primary antibody. Immunogold labelling with 15-nm particles was almost exclusively found overlying dense core vesicles (dcvs) and preabsorption of the primary antibody with synthetic GnRH eliminated this labelling. Morphometric analysis was performed on tissue from two monkeys. Four types of profiles containing GnRH immunoactive dcvs were observed. Type I profiles were morphologically unremarkable with a cross sectional area of approximately 0.6 microm2 and probably represent intervaricose axon segments. Type II profiles, which were nominally larger than Type I structures, were characterized by a high density of round microvesicles, which were frequently concentrated along the neuronal membrane to form 'synaptoid' contacts with adjacent glia. Two additional and large GnRH profiles (>5 microm2) were observed. One (Type III) contained a high density of dcvs and mitochondria, and was considered analogous to an axonal swelling or Herring body in the magnocellular hypothalamo-neurohypophysial system. The Type IV structure, which was considered not to be a Herring body because of the relative low density of mitochondria was innervated by a classical symmetrical synapse. The functional significance of these observations is discussed.

Immunohistochemical study of T lymphocyte subsets on frozen substituted plastic embedding section of the bone marrow from patients with myelodysplastic syndrome and its clinical implication.

An immunohistochemical study of T lymphocyte subsets on frozen substituted plastic embedding bone marrow sections obtained from 10 patients with myelodysplastic syndrome (MDS) was presented. The results of qualitative and quantitative immunohistochemical analysis are as follows: (1) Labile antigens of T lymphocytes were well preserved, thus allowing analysis of distribution of T lymphocyte subsets in situ: (2) the average number of T3, T4 and T8 lymphocyte of the diffuse infiltrate was about 2%, 0.4%, 0.5%, respectively, of all nucleated cells in bone marrow, and T4/T8 of T cells were below 1.0 in patients with MDS; (3) there were cases of RAS showing T lymphocyte aggregation in bone marrow, but no patient exhibited progressive refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEBT). These findings indicated that the immunological abnormalities are of importance in the evaluation of pathogenesis and prognosis of MDS.

Image Analysis and Fourier Transform Infrared Light Microscopy and Transmission Electron Microscopy of Mercerized Cotton Yarns

Abstract Renewed interest in mercerization as a pre-treatment for textile finishing has led to research on the effects of temperature on this process. Mercerization, the swelling of cotton in caustic soda, causes changes in crystallinity from a Cellulose I structure to a Cellulose II structure, along with changes in fiber physical dimensions, increased dyeability, luster and tensile strength. These changes are differentiated by light microscopy, image analysis and FT-IR, and transmission electron microscopy. Cotton yarns were mercerized with an aqueous solution of 23% NaOH and 1% wetting agent. The yarns were treated for 1 hour at 0, 20, 60, 80, and 110° C, and washed in distilled water at the same temperature as the treatment bath. Rapid embeddment procedures have been developed for analyzing the effects of treatments on cotton fiber structure. Cotton fiber cross-sections were prepared for light microscopy/image analysis by encasing the fibers in a tube,1 embedding in mefhacrylate plastic (Fig. la), polymerizing for 30 minutes with UV, and sectioning.

Cryo‐Immunogold Electron Microscopy

AbstractThis unit describes subcellular localization of proteins/antigens using high‐resolution cryo‐immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, since aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar‐coated grids.

The use of histology in the study of plant tissue culture systems—Some practical comments

Histological methods have contributed significantly to our understanding of in vitro culture systems. A good histological study based on anatomical and histochemical changes provides insight into cellular processes and provides clues that allow for the proposal of hypotheses for further experimentation. This article serves to draw attention to the use of a histological approach to one’s experimental system. Some of the common mistakes in the handling and processing of explants are discussed. A protocol for the plastic embedding method is detailed.

Rethinking Casein Micelle Structure Using Electron Microscopy

Numerous models for the structure of casein micelles have been proposed in the past. Based on the chemical and physical properties of micelles, these models fall into two general categories, models of internal structure and models of submicelles. Electron microscopy has been used to provide evidence for these models, although the available techniques imposed limits. With the development of newer electron microscopes, the structure of the bovine casein micelle has been further examined. Even at high resolution, scanning electron microscopy does not produce satisfactory images of casein micelles because the application of metal coatings (2 to 10nm thickness) obscures the proteins from view, and the resultant images of casein micelles resemble a lumpy sphere. Transmission electron microscopy has been used with varying results. Freeze-fracture replica methods coat the fine protein structure with metal, as occurs with scanning electron microscopy. Negative staining that includes air-drying introduces artifacts into the sample during drying. Plastic embedding can yield fairly representative images, but not without dimensional compression and some chemical modification. These chemical modifications occur predominantly from 1) chemical fixation, causing crosslinking of protein and thus shifting of their spatial resolution; 2) ethanol, causing coagulation of the proteins during dehydration; and 3) protein loss during infiltration with plastic. The inability to avoid these problems in the past has limited the ability to provide an accurate image of casein micelles. In this study, a cryopreparation technique has been applied to examine single casein micelles while retaining the maximum in situ structure. To avoid problems of metal coating and chemical modifications, casein micelles were absorbed to parlodion-coated grids, immersed in uranyl oxalate counterstain, quick frozen, and freeze-dried. This method yields images of whole, spherical casein micelles and shows the fine structural detail of the micellar proteins. Stereo images showed that the largest subaggregate particles within the casein micelle matrix were approximately 8 to 10nm in size.

A method for determining stress ratio of fatigue loading from the width and height of striation

In order to predict service load from fatigue fracture surface, the use of striation spacing ( s) and the ratio of striation height ( H) to s, H/ s is proposed. It is well known that s is correlated with the crack growth rate da/ dN. However, since the crack growth curve da/ dN–Δ K depends on the stress ratio R (= P min/ P max= K min/ K max), it is not possible to determine the minimum service load P min and the maximum service load P max only from the da/ DN–Δ K or s–Δ K relationship. In the previous paper, the authors found that there existed one-to-one correspondence between H/ s and the stress ratio R for 2017-T4 aluminum alloy and SUS304. Thus, the key to evaluate the stress ratio R in service load is an accurate determination of H/ s. The method used in the previous paper to measure H of striations is composed of several procedures such as cutting, embedding in plastics and finally sectioning specimens by a microtome (a special cutting machine). In this paper, an improved method of sectioning the fracture surface directly by the microtome is developed. By the new method, an increased number of accurate data of H/ s become available and the histogram indicating the scatter of H/ s values specific to a definite R ratio can be obtained. Three materials, 2017-T4 aluminum alloy, SUS304 and a high Mn steel, were investigated in order to study the effect of material properties on the relationship between H/ s and R. It was found that the mean value of the upper 20 data of 100 measured values of H/ s was a unique function of R for all three materials. This result implies, with a high degree of plausibility, the existence of one-to-one correspondence between H/ s and R regardless of materials. Thus, the master curve ( H/ s– R relationship) obtained in this study may be used for the failure analysis of various structures of different materials.

An En Bloc Staining Protocol Improves The Preservation of Lamellar Bodies in Alveolar Type II Epithelial Cells for Transgenic Mice Expressing Modified Pulmonary Surfactant Protein B

Abstract The extraction of lipids and phospholipids during dehydration and plastic embedding steps results in poor preservation of the phospholipid rich lamellar bodies (LB) in alveolar type II epithelial cells. To achieve better retention of phospholipids, we combined inflation fixation and an en bloc staining protocol using 4% aqueous uranyl acetate (UA), thereby improving the preservation of the LBs for both the wild type and transgenic mice expressing modified pulmonary surfactant protein B (SP-B; Akinbi et al., 1997). Lungs of 6-8 week-old mice were inflation fixed (Bunkingham and Weyder, 1981) with ice cold 2% paraformaldehye and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (SCB), pH 7.3, postfixed in fresh fixative at 4 °C overnight, incubated with 1% osmium tetroxide in 0.1 M SCB at room temperature for 2 hours, and stained en bloc with 4% aqueous UA overnight.

Neutrophils infiltrating bronchial epithelium in chronic obstructive pulmonary disease

In order to characterize neutrophil and eosinophil presence in the airways of patients with chronic obstructive pulmonary disease (COPD), bronchoscopy with bronchial washings and bronchial biopsies was performed in 12 smoking stable COPD subjects and 18 normal non-smoking control subjects. Bronchial biopsies were examined by light microscopy using plastic embedding and histochemical techniques to identify different cell types. Bronchial washing fluid of COPD patients was characterized by a predominance of neutrophils ( P=0·001), and a slight, but significant ( P=0·03), increase of eosinophil fraction. Subjects with COPD had higher number of neutrophils in the epithelium ( P=0·01), and eosinophils in the lamina propria ( P=0·01) than did control subjects. The thickness of reticular basement membrane was increased for COPD patients in comparison to control subjects ( P=0·01). The present study provides evidence of neutrophil infiltration both in bronchial washing and bronchial epithelium of patients with COPD, suggesting that the source of neutrophils in airway lumen may be the bronchial mucosa. Although less common than in asthma, airways of COPD subjects reveal eosinophil presence and airway remodelling.

Application of Confocal Laser Scanning Microscopy in Bone

The optical sectioning method of confocal laser scanning microscopy provides higher resolution than standard light microscope techniques. The use of optical rather than physical sections for detailed histological analyses of bone obviates the need for either decalcification or complex plastic embedding processes which are required as a routine for the preparation of thin microtome sections. In this study we have used confocal laser scanning microscopy for the morphological analyses of fresh unembedding human cortical bone, bone allograft and bone cement interfaces. Our results have indicated that such an approach has provided a relatively easy and rapid means for the assessment of the histology of normal and pathological bone.