Immunogold-Silver Staining Method for Lymphocyte Cell Surface Antigens with Light,Transmission Electron,and Scanning Electron Microscopy

Abstract

The immunogold-silver staining (IGSS) method combined with light, transmission electron, and scanning electron microscopy (LM, TEM and SEM, respectively) was used for detecting lymphocyte surface antigens. Two different sizes of colloidal gold particles (5 nrn and 15 nm) were applied as markers and IntenSEII kit as a physical developer for gold particles.

The silver enhanced gold particles were clearly observed on cell surfaces as black dots in LM and TEM and as white dots in SEM equipped with a mixed signal of secondary electron and back-scattered electron (SE/BE) signals. Monoclonal antibody (MAb)-positive cells possessing the complexes on their well preserved surfaces were easily identified among other lymphoid cells at low magnifications of LM or SEM equipped with SE/BE signals. Thus, the IGSS method has a great advantage for a qualitative screening such as the percentage of lymphocyte subsets in a cell suspension. However, the IGSS method was inadequate for semiquantitative study with antigen density on cell surfaces because gold particles enhanced with the physical developer were considerably enlarged, and a silver-gold complex was not considered to show one antigen site on cell surface. (The J Histotechnol 16:217, 1993)     

Simultaneous processing of substrate-dependent monolayer cultures for scanning and transmission electron microscopy

Summary A reliable method is described for processing substrate-dependent cells raised on culture-grade plastic for both scanning (SEM) and transmission electron microscopy (TEM). This technique allows collection of specimens for TEM and SEM from the same culture dish or flask. In this way it is possible to study the surface morphology (SEM) and thin section ultrastructure of cells from contiguous regions of a culture. Specific regions of a culture can be selected and processed so that specimens retain orientation throughout mounting or embedment and sectioning. The method is applicable both to confluent cultures as well as isolated colonies.     

Correlative Light Optical,Scanning Electron,and Transmission Electron Microscopy of Skeletal Muscle in Muscular Dystrophy and Muscular Atrophy: A Pilot Study

Biopsies of skeletal muscle from three different cases of muscular dystrophy and one case of spinal muscular atrophy that had been fixed with Karnovsky's fluid were either routinely prepared for scanning electron microscopy (SEM) or were frozen to-20°C and sectioned on a steel knife in a cryostat at 5-10 μm. The sections were coverslipped and examined using a light microscope equipped with polarizing optics (Pol). After areas were selected, the sections were prepared for SEM and thereby examined. The tissues on the slides that had been observed with light microscopy (LM) and SEM were prepared further for transmission electron microscopy (TEM) by infiltrating them with Epon and cutting sections at approximately 100 nm on an ultramicrotome. It is shown that the stage of contraction in one pathologic myofiber may vary along its length. The following advantages may be realized by using correlative (Pol → SEM → TEM) microscopy on skeletal muscle biopsies: 1) lesions can be differentiated from “normal” surrounding tissue; 2) doubtful structures can be reexamined with the SEM and TEM; and 3) the SEM image of different states of muscle contraction can be reinterpreted in the light of the Pol or TEM image.     

Correlative Light Optical, Scanning Electron, and Transmission Electron Microscopy of Skeletal Muscle in Muscular Dystrophy and Muscular Atrophy: A Pilot Study

Biopsies of skeletal muscle from three different cases of muscular dystrophy and one case of spinal muscular atrophy that had been fixed with Karnovsky's fluid were either routinely prepared for scanning electron microscopy (SEM) or were frozen to-20°C and sectioned on a steel knife in a cryostat at 5-10 μm. The sections were coverslipped and examined using a light microscope equipped with polarizing optics (Pol). After areas were selected, the sections were prepared for SEM and thereby examined. The tissues on the slides that had been observed with light microscopy (LM) and SEM were prepared further for transmission electron microscopy (TEM) by infiltrating them with Epon and cutting sections at approximately 100 nm on an ultramicrotome. It is shown that the stage of contraction in one pathologic myofiber may vary along its length. The following advantages may be realized by using correlative (Pol → SEM → TEM) microscopy on skeletal muscle biopsies: 1) lesions can be differentiated from “normal” surrounding tissue; 2) doubtful structures can be reexamined with the SEM and TEM; and 3) the SEM image of different states of muscle contraction can be reinterpreted in the light of the Pol or TEM image.     

Anatomy of the cecum of the laboratory mouse and rat

Summary The anatomy of the cecum of the laboratory mouse and rat was studied from a comparative standpoint. The topographical situation, mesenterial connections and arterial supply to the ceca were examined macroscopically. Dried specimens were made to study the morphological form and internal structures. Microscopically, at the light, transmission and scanning electron microscopic levels, the characteristic structural aspects of the ceca of both species were observed.The mucosa of the mouse cecum is arranged in soft-contoured, looped configurations (SEM), which appear as wide-opened crypts light microscopically. The rat has a similar appearance except that the configurations are more densely arranged and the crypts narrower. With TEM, the cecal epithelial cells of both species display typical features of intestinal absorptive cells. The observations made in the rat and mouse are compared with eachother and with other species where detailed information is available.     

体外培养人表皮黑素细胞样本制备及超微结构观察

目的探讨体外培养正常人表皮黑素细胞(melanocytes,MC)透射电镜制样方法及超微结构观察的作用。方法采用M254 MC培养基及添加剂HMGS培养和纯化人表皮MC;分别采用常规制样方法和以PET聚酯薄膜为培养细胞的支持物制备透射电镜样本。结果人表皮MC透射电镜观察发现,常规制样标本可观察人表皮MC及其树状突起横切面的超微结构。PET聚酯薄膜作为培养MC支持物的样本可观察人表皮MC和树状突起的完整超微结构及树状突起的纵切面超微结构。结论 PET聚酯薄膜可作为人表皮MC体外培养良好的支持材料。应用不同样本制备方法可以观察MC的超微结构,使用PET聚酯薄膜作为细胞支持物制备的标本更利于MC黑素体代谢和转移的观察。     

Ultrastructural investigation of Zimmermann-Laband syndrome

Zimmermann-Laband syndrome (ZLS) is a very rare autosomal dominant inherited condition characterized by 3 major clinical findings of which gingival hyperplasia are always present. The great heterogenicity of the syndrome is illustrated by the numerous variable clinical findings described in the literature. The purpose of the study was to examine a patient diagnosed with ZLS and to describe possible new characteristics of this rare syndrome, including the ultrastructural morphology using a transmission electron microscope (TEM) of the gingival and dermal fibroblasts. The ultrastrucutral morphology as has not previously been described in the literature. Tissue was collected from the alveolar ridge and skin of the forearm for TEM. TEM studies indicated the presence of prominent fibroblasts situated among numerous regular dense connective tissue bundles. Genetic analysis showed a new chromosomal insertion, ins(12;8)(p11.2;q11.2q24.3), suggesting that the gene responsible for the syndrome lies on chromosome 8.     

Application of electron microscopy to the study of structural changes in the liver in non-A,non-B hepatitis

Ultrastructural studies employing techniques such as alternative electron metal stain, high-angle tilting and high-voltage electron microscopy were carried out on liver biopsies obtained from chimpanzees infected with non-A, non-B hepatitis. Typical derangement of the endoplasmic reticulum leading to the formation of tubular structures in hepatocytes was observed. The use of potassium permanganate as an alternative stain revealed two features which have not been previously described. The first of these shows the wall of the tubular structures to be composed of a well-defined fibrillarlike meshwork with a periodicity of approximately 15 nm. The second feature is the demonstration of clusters of fibrin-like inclusions consisting of striated fibrils in the neighbourhood of the tubular structures. The presence of intracytoplasmic fibrin may indicate non-specific morphological evidence of cell injury. Crystalline structures containing arrays of particles with an average size of 24 nm were also observed in the endoplasmic reticulum of endothelial cells of the hepatic sinusoids. Morphological differences between the crystalline lattice and the reticular arrangement, demonstrated with the use of high-angle tilting of the specimen in the electron microscope suggest that the arrays may not be viral particles but a reflection of pathological response of the host cell.     

The morphology of human immunodeficiency virus (HIV) by negative staining

We have examined preparations of human immunodeficiency virus 1 (HIV-1) and HIV-2 by negative staining electron microscopy. HIV-2 cultures contained large numbers of 130-200 nm particles containing a 130-nm-long by 30-70 nm-wide core. This core is probably of conical or pear-shaped morphology. Some particles exhibited a short fringe that could be seen to comprise a regular arrangement of repeating subunits when visualised end on. Identical particles were found in HIV-1 cultures but in much lower numbers. Attempts to carry out negative staining immune EM were unsuccessful. Also detected in both HIV-1 and HIV-2 cultures were small (70-80 nm) fringed viruslike particles. The possible significance of these particles is discussed.     

Decreased protein and gene expression of hepatic cholesterol 7a-hydroxylase associated with dilated endoplasmic reticulum in chronic hypothyroid rats

Chronic hypothyroidism is associated with an increased risk for cardiovascular disease attributed, in part, to increased serum cholesterol and atherosclerosis. Decreased hepatic activity of cholesterol 7a-hydroxylase (CYP7A1) is thought to contribute to hypercholesterolemia in hypothyroidism. The endoplasmic reticulum (ER) has been shown to be a central organelle, and it is the location of CYP7A1 in hepatocyte. The aim of the present study was to determine whether the expression of CYP7A1 mRNA and protein could be decreased and whether ER could undergo morphological changes in a rodent model of chronic hypothyroidism induced by methimazole (MMI). Male Wistar rats were treated with MMI (0.04% wt/vol) or regular water for 14 weeks. Hepatic CYP7A1 mRNA was analyzed on quantitative real-time polymerase chain reaction (PCR). Hepatic CYP7A1 protein expression was assessed on western blot. In conjunction with these molecular biological assessments the morphology of ER was evaluated on transmission electron microscopy (TEM). Serum total cholesterol increased significantly compared to controls and was associated with decreased CYP7A1 mRNA and protein, and TEM indicated remarkable dilation of ER in hepatocytes in the chronic hypothyroid rats. These findings suggest that a relationship between the decreased expression of hepatic CYP7A1 mRNA and protein and dilated ER could exist, which may contribute to hypercholesterolemia in chronic hypothyroidism.     

The molecular mass of adenovirus type 5 as determined by means of scanning transmission electron microscopy (STEM)

The mass of adenovirus type 5 was determined by means of computer-assisted scanning transmission electron microscopy (STEM). Arithmetic mean of 157 +/- 10(SD) X 10(6) daltons and mode between 160 and 170 X 10(6) daltons compare favourably with previously reported data. The advantages of the STEM-procedure over the physical and chemical techniques are: low amounts of purified virus particles are needed; visual control of the physical state of virus particles; no need to know the chemical composition or protein concentration of the virus sample.     

Ultrastructural study of Helicobacter pylori-associated gastritis

Endoscopic biopsies of antral mucosa from 26 patients with Helicobacter pylori-associated gastritis were studied by electron microscopy (EM). Scanning electron microscopy (SEM) showed clustering of H. pylori in the intercellular areas, being entrapped by the microvilli which were decreased at the sites where the bacilli were seen. The observations of SEM were confirmed by transmission electron microscopy (TEM), which showed adherence of the bacilli to the cell surface, producing cup-shaped depressions in the epithelial cells, and occasionally intracellular infiltration by H. pylori. There were also depletion of mucus granules, degenerative changes, and disruption of intercellular junction complexes of the epithelial cells. Post-treatment biopsies showed complete disappearance of the bacilli, and ultrastructural changes associated with H. pylori infection were resolved.     

Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety

Transmission electron microscopy (TEM) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer‐scale resolution. Between the 1960s and 1990s, TEM contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. The diagnosis of viral infections improved considerably during the 1990s, with the advent of highly sensitive techniques, such as enzyme‐linked immunosorbent assay (ELISA) and PCR, rendering TEM obsolete for this purpose. However, the last 20 years have demonstrated the utility of this technique in particular situations, due to its “catch‐all” nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. Thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, TEM provided the answer. TEM is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. It is also used to check the microbiological safety of biological products. Many biopharmaceuticals are produced in animal cells that might contain little‐known, difficult‐to‐detect viruses. In this context, the “catch‐all” properties of TEM make it possible to document the presence of viruses or virus‐like particles in these products.     

Ultrastructural features in chronic non-A, non-B (NANB) hepatitis: A controlled blind study

Liver biopsies from 12 patients with chronic Non-A, Non-B (NANB) hepatitis, 7 with hepatitis B surface antigen (HBsAg) positive chronic liver disease, 1 HBsAg positive normal carrier, and 4 patients with non-viral liver disease, were examined by electron microscopy for cytoplasmic and nuclear changes. Aggregates of particles measuring 20-35 nm in diameter were noted in the nuclei of 8 of 12 patients with NANB chronic hepatitis, but not in the other groups. The tubular changes seen in the endoplasmic reticulum (ER) of chimpanzees with NANB hepatitis were not noted in biopsies from any of our patients.     

Melatonin release by exocytosis in the rat parotid gland

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non‐protein‐bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the β‐adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg^?1), the right parotid gland was removed; pre‐administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty‐six per cent of the total granular population (per 100 μm² per cell area) displayed melatonin labelling in the matrix; three‐quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so‐called regulated secretory pathway. During its stay in granules, anti‐oxidant melatonin may protect their protein/peptide constituents from damage.     

Tunable Morphologies From Bulk Self‐Assemblies of Poly(acryloxypropyl triethoxysilane)‐b‐poly(styrene)‐b‐poly(acryloxypropyl triethoxysilane) Triblock Copolymers

Reactive poly(acryloxypropyl triethoxysilane)‐b‐poly(styrene)‐b‐poly(acryloxypropyl triethoxysilane) (PAPTES‐b‐PS‐b‐PAPTES) triblock copolymers are prepared through nitroxide‐mediated polymerization (NMP). The bulk morphologies formed by this class of copolymers cast into films are examined by small‐angle X‐ray scattering (SAXS) and transmission electron microscopy (TEM). The films morphology can be tuned from spherical structures to lamellar structures by increasing the volume fraction of PS in the copolymer. Thermal annealing at temperatures above 100 °C provides sufficient PS mobility to improve ordering.     

Imaging of Polymeric Nanoparticles: Hard Challenge for Soft Objects

With the emergence of nanotechnology and nanoscience in the past two decades a thorough characterization at the nanoscale became more and more important. The characterization of nanoparticles is the main issue for the understanding of their properties. Besides averaging methods like scattering techniques the direct imaging of nanoparticles by microscopy methods is the major characterization method. Especially electron microscopy proved to be a valuable tool to obtain morphological and analytical information. The aim of this article is to point out the prospects as well as the pitfalls of this technique with special emphasis on the electron microscopical imaging of polymeric nanoparticles. We will present two alternative methods to cryo‐TEM preparation: embedding the sample into an ultrathin film of trehalose as already used for the preparation for biological samples and the preparation of an ultrathin, free standing ionic liquid film as embedding matrix for nanoparticular structures.

     

Comparative studies of the same adenocarcinoma cells,macrophages, and mesothelial cells by light microscopy,scanning electron microscopy,and transmission electron microscopy

The identification of cells in body cavities of cancer patients is sometimes difficult to make. In order to make a definite cytological diagnosis, we observed the same cells by using light microscopy (LM)-scanning electron microscopy (SEM)-transmission electron microscopy (TEM). In this study we first stained cells by the Papanicolaou method after fixation in 1% glutaraldehyde for LM, and then attempted to observe them successively by SEM-TEM after fixation in 1% paraformaldehyde and 1.25% O^s0⁴. Our method and procedures in examining successively one and the same cells in body cavity fluids by using LM, SEM, and TEM ensured accurate discrimination among adenocarcinoma cells, mesothelial cells, and macrophages. The results of this study suggest that LM-SEM-TEM may be of diagnostic value in distinguishing among mesothelial cells, macrophages, and adenocarcinoma cells. This method also succeeded in disclosing differences between the ultrastructure of the cell surfaces, and those of the cytoplasm, and of the nuclei It is desirable that LM-SEM-TEM observation can be introduced into various aspects in order to obtain an improvement in the diagnosis by cytologic examination, the judgment of therapeutical effects, drug selection, and prognostic presumption. Diagn Cytopathol 1994; 11:333–342. © 1994 Wiley-Liss, Inc.