scholarly journals Electron Microscopy of In-Plaque Phage T3 Assembly: Proposed Analogs of Neurodegenerative Disease Triggers

2020 ◽  
Vol 13 (1) ◽  
pp. 18 ◽  
Author(s):  
Philip Serwer ◽  
Barbara Hunter ◽  
Elena T. Wright
Keyword(s):  
Electron Microscopy ◽  
Innate Immunity ◽  
Neurodegenerative Disease ◽  
Virus Assembly ◽  
Uranyl Acetate ◽  
Host Responses ◽  
Thin Sections ◽  
Viral Capsids ◽  
Acetate Lead ◽  
Forming Temperature

Increased knowledge of virus assembly-generated particles is needed for understanding both virus assembly and host responses to virus infection. Here, we use a phage T3 model and perform electron microscopy (EM) of thin sections (EM-TS) of gel-supported T3 plaques formed at 30 °C. After uranyl acetate/lead staining, we observe intracellular black particles, some with a difficult-to-see capsid. Some black particles (called LBPs) are larger than phage particles. The LBP frequency is increased by including proflavine, a DNA packaging inhibitor, in the growth medium and increasing plaque-forming temperature to 37 °C. Acidic phosphotungstate-precipitate (A-PTA) staining causes LBP substitution by black rings (BRs) that have the size and shape expected of hyper-expanded capsid containers for LBP DNA. BRs are less frequent in liquid cultures, suggesting that hyper-expanded capsids evolved primarily for in-gel (e.g., in-biofilm) propagation. BR-specific A-PTA staining and other observations are explained by α-sheet intense structure of the major subunit of hyper-expanded capsids. We hypothesize that herpes virus triggering of neurodegenerative disease occurs via in-gel propagation-promoted (1) generation of α-sheet intense viral capsids and, in response, (2) host production of α-sheet intense, capsid-interactive, innate immunity amyloid protein that becomes toxic. We propose developing viruses that are therapeutic via detoxifying interaction with this innate immunity protein.

Ultrastructural differences in the exosporium of the Sterne and Vollum strains of Bacillus anthracis

1968 ◽  
Vol 14 (12) ◽  
pp. 1297-1299 ◽  
Author(s):  
M. J. Kramer ◽  
Ivan L. Roth
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Bacillus Anthracis ◽  
Potassium Permanganate ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Single Application ◽  
Thin Sections ◽  
Acetate Lead ◽  
Different Strains

Spores from three different strains of Bacillus anthracis were examined by electron microscopy for the presence of a hair-like nap previously reported to be present on the exosporium of spores of the Sterne strain (avirulent). In addition to that strain, the Vollum strain (virulent) and a rough, avimlent variant of the Vollum were utilized in the current studies.Spores were fixed with Kellenberger's standard OsO4 fixative and embedded in Maraglas. Thin sections were poststained with various combinations of the following: potassium permanganate, uranyl acetate, lead citrate. The nap on the exosporium of spores of the Sterne strain was revealed most clearly when thin sections were poststained with all of the aforementioned stains. Post-staining by a single application of any of the three reagents resulted in a nap that was barely perceptible.The surface of the exosporium of spores from the Vollum strain and the rough, avirulent variant was found to be quite different from that of the Sterne strain. On the two former, the surface layer is approximately one-third as thick as the layer of hairs in the nap on the latter.

Ultrahistochemical Analysis of the Chick Embryo Cornea Using A Non-Dispersive X-Ray Detector

1972 ◽  
Vol 30 ◽  
pp. 404-405
Author(s):  
T. Ohkura ◽  
M. Takashio ◽  
T. Watanabe
Keyword(s):  
Electron Microscopy ◽  
Chick Embryo ◽  
Alcian Blue ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Thin Sections ◽  
X Ray ◽  
Transmission Electron ◽  
Glutaraldehyde Solution ◽  
Tissue Specimens

The experiments which we report here were performed to show qualitatively the presence or abscence of Cu in the mesostroma of the chick embryo cornea stained with alcian blue 8GS. Strips of the cornea were fixed in a buffered glutaraldehyde solution (2.5%, pH 7.4) and coloured with alcian blue 8GS at pH 2.5. Tissue specimens were postosmicated, dehydrated and embedded in Epon. Tissue blocks for the conventional transmission electron microscopy were prepared without alcian blue treatment, and the thin sections were stained with uranyl acetate and/or lead citrate. Fig. la shows the mesostroma of 3rd day chick embryo cornea; filaments run in various directions, and reveal no visible periodicity. Interfibrillar substances can not be demonstrated by the conventional method of the electron staining. The alcian blue treatment reveals the interfibrillar substances, which stand out in contrast as shown in Fig. lb.

Uranyl acetate staining under different conditions of preparation, storage and use

1990 ◽  
Vol 48 (3) ◽  
pp. 726-727
Author(s):  
R.H.M. Cross ◽  
A.N. Hodgson ◽  
R.T.F. Bernard
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Biological Tissue ◽  
Storage Time ◽  
Uranyl Acetate ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Standard Methods ◽  
Gut Epithelium ◽  
Transmission Electron

Uranyl acetate is routinely used in the staining of thin sections of biological tissue for transmission electron microscopy. Although many methods for its preparation and use have been described, there is seldom reference to the reasons for variations in concentration, solvent, storage time and staining time. Likewise, possible variations in the effects of staining under different conditions are largely ignored. In order to gain clarity on this issue an attempt has been made to test three variables (solvent, storage time and use in light or dark) under controlled experimental conditions.The tissues used for the experiment were the testis of a marine limpet, the gut epithelium of a fresh-water catfish, and the kidney of a rat; all of which were fixed and embedded by standard methods. The uranyl acetate solutions were prepared at the outset of the experiment and dispensed into small volumes and stored in the dark at 4°C until required.

Electron Microscopy as an aid to diagnosis in pediatric hematology/oncology

1989 ◽  
Vol 47 ◽  
pp. 1062-1063
Author(s):  
K. L. Saving ◽  
R. C. Caughey
Keyword(s):  
Electron Microscopy ◽  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Thin Sections ◽  
Megakaryoblastic Leukemia ◽  
Pediatric Hematology ◽  
Transmission Electron ◽  
Microscopy Techniques

This presentation is designed to demonstrate how scanning and transmission electron microscopy techniques can be utilized to confirm or support a variety of unusual pediatric hematologic/oncologic disorders. Patients with the following diagnoses will be presented: (1) hereditary pyropoikilocytosis, (2) familial erythrophagocytic lymphohistiocytosis, (3) acute megakaryoblastic leukemia, and (4) pseudo-von Willebrand’s disease.All transmission and scanning electron microscopy samples were fixed in 2.5% glutaraldehyde, rinsed in Millonig’s phosphate buffer, and post-fixed with 1% osmium tetroxide. The transmission samples were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole ’ s non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin. Ultramicrotomy thin sections were stained with uranyl acetate and lead citrate and scanned using a JEOL-JEM 100C, The scanning samples were dehydrated with graded series of ethanols, critical point dried with CO2, gold-coated, and scanned using a JEOL-JSM 35. The peroxidase samples were fixed in 3% glutaraldehyde, incubated in diaminobenzidine (DAB), dehydrated with ethanol, embedded with Epon 812, and scanned without post-staining using a JEOL-JEM 100C.

EM Study of Isopod Hemocytes

1998 ◽  
Vol 4 (S2) ◽  
pp. 1138-1139
Author(s):  
G. M. Vernon ◽  
E. J. Rappa ◽  
W. C. Murray ◽  
R. Witkus
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Morphological Analysis ◽  
Standard Technique ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Small Granule ◽  
Thin Sections ◽  
Cytoplasmic Granules ◽  
Transmission Electron

Crustacean hemocytes have been characterized on the basis of cell size and nature of cytoplasmic granules. Based on light microscopic morphological analysis and cytochemistry, investigators variously named the hemocyte types (agranular, small-granule, large granule, undifferentiated, hyaline cells, non-explosive, explosive granulocytes, etc.). In his study of the isopod Armadillidium vulgare Faso adopted the terminology of Benjamin and James and referred to the hemocytes as hyaline cells, semi-granulocytes and granulocytes.In the present investigation we have studied the hemocytes of two isopods, Oniscus asellus and Armadillidium nasatum, using transmission electron microscopy. Hemolymph was collected by penetrating the posterior dorsal exoskeleton of 20 animals of each genus with a microcapillary pipette and drawing 3-5μL per isopod. The samples were processed following a standard technique. Thin sections were collected on 300 mesh copper grids, counterstained with 2% aqueous uranyl acetate and lead citrate, and viewed with a JEOL 1010 electron microscope.

Computer-assisted analysis of regenerating rabbit retinal pigment epithelium

1990 ◽  
Vol 48 (3) ◽  
pp. 408-409
Author(s):  
G. E. Korte ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Uranyl Acetate ◽  
Computer Assisted ◽  
Thin Sections ◽  
Transmission Electron ◽  
Assisted Analysis ◽  
Routine Procedures

While examining thin sections of regenerating rabbit retinal pigment epithelium (RPE) we observed profound changes in ultrastructure as the cells matured. To assist in studying this problem serial thick sections (0.25 μm) through regenerating RPE cells were studied by highvoltage electron microscopy (HVEM) and a computer program (STERECON) used to generate stereo reconstructions.Tissue was obtained from rabbits that received sodium iodate iv -- which destroys large expanses of RPE and permits examination of regeneration from surviving cells. One-two weeks after iodate administration the rabbits were euthanized, the eyes removed and tissue processed for transmission electron microscopy by routine procedures. Serial sections 0.25 μm thick were mounted on Formvar coated slot grids, stained with uranyl acetate and lead citrate and examined with an AEI EM7 1.2 MV HVEM at 1000 kV. Figure 1 illustrates observations made on six sections through the cell seen in Fig. 2. The cell was photographed at 5000x and printed as a montage at 15,000x.

First Report of a Putative Cyanophage, Mc-1, of Microcoleus Sp.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1142-1143
Author(s):  
J. R. Rosowski ◽  
J. J. Shaffer ◽  
E. L. Martin ◽  
T. A. Kokjohn ◽  
K. W. Lee
Keyword(s):  
Electron Microscopy ◽  
Water Medium ◽  
Filament Formation ◽  
Thin Sections ◽  
Viral Capsids ◽  
Blue Green Algae ◽  
Current Review ◽  
Transmission Electron ◽  
A Current ◽  
Mucilage Secretion

A current review (1) lists 21 cyanophages of coccoid and filamentous heterocystic and non-heterocystic blue-green algae (BGA). The capsid diameters of these viruses range from 50-90 nm, and none have been previously reported for Microcoleus,a sheath-producing, filamentous, surface colonizing BGA (Fig. 1), of the order Oscillatoriales, and class Cyanophyceae (= Cyanobacteria). From a freshwater culture of Microcoleussp. we report here a putative cyanophage with a side-to-side hexagonal capsid diameter of 140 nm, based on measurements from thin sections of algal filaments prepared for transmission electron microscopy (TEM).An earlier report (2) documented the ultrastructure of mucilage secretion, and its role in sheath, trichome and filament formation in an isolate of the BGA Microcoleus sp. grown in soil-water medium. Now we report large cyanophage-like particles in a majority of cells making up the trichomes. These putative hexagonal viral capsids (Figs. 2-5) are mainly clustered in the center or to one side of the nucleoplasm.

scholarly journals Optimization of Protocols for Pre-Embedding Immunogold Electron Microscopy of Neurons in Cell Cultures and Brains

2021 ◽  
Author(s):  
Jung-Hwa Tao-Cheng ◽  
Virginia Crocker ◽  
Sandra Lara Moreira ◽  
Rita Azzam
Keyword(s):  
Electron Microscopy ◽  
Cell Cultures ◽  
Uranyl Acetate ◽  
Immunogold Electron Microscopy ◽  
En Bloc ◽  
Thin Sections ◽  
Optimal Protocol ◽  
Small Gold Particle ◽  
Specific Proteins ◽  
Secondary Antibodies

Abstract Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

Unusual Inclusions in Neutrophils of two Human Renal Allografts

1976 ◽  
Vol 34 ◽  
pp. 242-243
Author(s):  
R. Valenzuela ◽  
S.D. Deodhar ◽  
D.G. Osborne ◽  
W.E. Braun ◽  
L.H.W. Banowsky
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Renal Allograft ◽  
Light And Electron Microscopy ◽  
Microscopy Study ◽  
Uranyl Acetate ◽  
Electron Microscopy Study ◽  
Thin Sections ◽  
Blood Disorder ◽  
Cadaver Donor

During a retrospective light and electron microscopy study of 158 human renal allograft biopsies, we observed, in two cases, unusual inclusions in the neutrophils trapped in glomeruli and paratubular capillaries. For electron microscopy, the specimens were fixed in glutaraldehyde, post-fixed in osmium tetroxide and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate. Eighty-six of the electron microscopically examined biopsies did not contain any neutrophils for evaluation, and only 10 biopsies showed four or more neutrophils.The two patients, A age 35 and B age 42, had received a cadaver donor renal allograft because of chronic renal failure secondary to hereditary nephritis and nephrosclerosis respectively. Neither patient was affected by any known primary blood disorder.

Morphometric comparison of uranyl acetate/lead citrate-stained and peroxidase-stained neutrophil granules

1990 ◽  
Vol 48 (3) ◽  
pp. 756-757
Author(s):  
Charles S. Gilbert ◽  
Richard T. Parmley ◽  
Joseph M. Kinkade
Keyword(s):  
Human Peripheral Blood ◽  
Uranyl Acetate ◽  
Buffy Coat ◽  
Significant Heterogeneity ◽  
Lead Citrate ◽  
Thin Sections ◽  
Low Viscosity ◽  
Physical Density ◽  
Acetate Lead ◽  
Blood Neutrophils

Although neutrophil granules can be broadly divided into peroxidase-positive and peroxidasenegative granules, recent studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. The present study was undertaken to evaluate this heterogeneity morphometrically. Human peripheral blood neutrophils were collected from normal volunteers in heparin or EDTA and fixed in 3% glutaraldehyde as a cell suspension or minced buffy coat. An aliquot was stained for myeloperoxidase using 3,3’-diaminobenzidine (DAB) as substrate. All samples were post fixed in 1% OSO4 and embedded in Spurr low viscosity medium. Additionally neutrophil granules isolated according to the method of Rice, et al. were similarly embedded. Thin sections of morphologic preparations were counterstained with methanolic uranyl acetate and aqueous lead citrate (UALC) (Fig. 1), whereas DAB stained specimens were counterstained with LC only (Fig. 2). Attempts to combine UALC and DAB staining in the same section obscured the density imparted by DAB. LC failed to impart significant density to DAB-negative granules in DAB-LC stained specimens resulting in an underestimation of this granule population.

Sign in / Sign up

Export Citation Format

Share Document