Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

scholarly journals Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology.

1979 ◽  
Vol 27 (10) ◽  
pp. 1337-1342 ◽  
Author(s):  
J Walton
Keyword(s):  
Electron Microscopy ◽  
Aspartic Acid ◽  
Lead Nitrate ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.

scholarly journals Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

1958 ◽  
Vol 4 (6) ◽  
pp. 711-716 ◽  
Author(s):  
Edward Essner ◽  
Alex B. Novikoff ◽  
Bertha Masek
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Function ◽  
Molecular Transport ◽  
Enzyme Reaction ◽  
Bile Canaliculus ◽  
Nucleotidase Activity ◽  
Reaction Products ◽  
Thin Sections

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.

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.

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.

Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds

1980 ◽  
Vol 42 (1) ◽  
pp. 291-304
Author(s):  
K.W. Adolph
Keyword(s):  
Electron Microscopy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nuclear Periphery ◽  
Sucrose Density Gradient ◽  
Rnase A ◽  
Micrococcal Nuclease ◽  
Order Structure ◽  
Dextran Sulphate ◽  
Thin Sections ◽  
Thin Sectioning

Histone-depleted nuclei were prepared from isolate HeLa nuclei by extracting the histones and other proteins with polyanions (dextran sulphate and heparin) or with high salt concentrations as used previously. The particles were characterized by sucrose density gradient sedimentation, thin sectioning and electron microscopy, and by polyacrylamide gel electrophoresis. The general result of the experiments is that the DNA in the histone-depleted nuclei is highly organized, and that this residual, higher-order structure is maintained by a reproducible subset of nuclear proteins, and perhaps by RNA. Furthermore, the residual proteins remain associated, in some conditions, as rapidly sedimenting structures even when the DNA is digested with nucleases. These nuclear scaffolds can resemble extracted nuclei. Histone-depleted HeLa nuclei sediment in sucrose density gradients as well defined peaks with sedimentation coefficients of around 12 000 S, when 2M NaCl is used to extract the histones, or 6 000 S, when dextran sulphate is used. The rate of sedimentation is drastically decreased by treating the particles with trypsin, and reduced to a lesser extent with RNase A. Thin sectioning and electron microscopy show that histone-depleted nuclei possess the nuclear periphery and that internal material is also present. These general features are also seen in thin sections of nuclear scaffolds, which are prepared by treating the nuclei with micrococcal nuclease of DNase I in addition to extracting the histones. Two groups of major proteins are associated with histone-depleted HeLa nuclei and the nuclear scaffolds: One group has molecular weights of 50 000-55 000 Daltons. The major species of this latter group of proteins have mobilities that are similar to the proteins of the metaphase chromosomal scaffold.

scholarly journals THE LOCALIZATION BY ELECTRON MICROSCOPY OF HELA CELL SURFACE ENZYMES SPLITTING ADENOSINE TRIPHOSPHATE

1963 ◽  
Vol 19 (2) ◽  
pp. 325-336 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Electron Microscopy ◽  
Enzyme Activity ◽  
Adenosine Triphosphate ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Membrane System ◽  
Thin Sections ◽  
A Cell ◽  
Dense Band ◽  
A Chain

Cultures of normally proliferating Hela cells have been examined in thin sections by electron microscopy following glutaraldehyde fixation, staining in Wachstein and Meisel's adenosine triphosphate containing medium, postosmication, and embedding in an epoxy resin. The cells were stained in suspension in order to ensure uniform accessibility to reagents. Discrete localization of the enzyme reaction product (lead phosphate) was found at the plasma membranes of about half the cells, but nowhere else. It appeared in the form of an intensely electron-opaque deposit lying close against the outer surface of the cells and varying in amount from a chain of small particles to a dense band about 30 mµ in width. This opaque reaction product was present over microvilli when absent elsewhere on a cell, was heaviest where microvilli and processes were profuse, and was minimal or lacking where cell surfaces were smooth. These observations are discussed in relation to both the idea that surface enzyme activity varies with the physiological phase of individual cells in a population, and the problem of how such enzyme activity becomes manifest at a given site on a morphologically changing membrane system.

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.

The Effect of Electron Microscopy on Pathological Diagnosis of Neoplasm

1990 ◽  
Vol 48 (3) ◽  
pp. 248-249
Author(s):  
Xiaojun Zhou ◽  
Taihe Zhang
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Frozen Section ◽  
Protein A ◽  
Functional Classification ◽  
Lead Citrate ◽  
Thin Sections ◽  
Tumor Tissues ◽  
Frozen Section Diagnosis

Although electron microscopy (EM) has contributed enormously to an understanding of the structural intricacies of tumor cells, the usefulness of EM in pathological diagnoses of neoplasms has not been readily appreciated by general pathologists. In the present study, 223 tumors submitted for EM diagnosis were analyzed in an attempt to gain further information concerning the contribution of EM to tumor diagnosis.223 neoplasms were submitted to EM for their final diagnoses when histopathological diagnoses were obscure, which represented about of the total number of surgical tumor specimens. Most specimens were taken at the time of frozen section diagnosis and a small number of tissues were originally fixed informaldehyde. All of tissues were fixed with buffered glutaradehyde, postfixed with osmium tetroxide and embedded in Epon 812. Ultrasections were made after semith in sections were examined to verify that representative tumor tissues were present. Thin sections were stained with uranium acetate and lead citrate, and examined under JEM-1200 EX electron microscope. In selected cases, mainly with neuroendocrine tumors, nickel grid-mounted sections were subjected to post embedding immunoelectron microscopy (IEM) using protein A-gold for more detailed functional classification. Protein A-gold probes were prepared as Wang and co-workers described.

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.

An Extemporaneous Lead Citrate Stain for Electron Microscopy

1967 ◽  
Vol 25 ◽  
pp. 148-149 ◽  
Author(s):  
Aly Fahmy
Keyword(s):  
Electron Microscopy ◽  
Sodium Hydroxide ◽  
Sodium Citrate ◽  
Lead Nitrate ◽  
Lead Citrate ◽  
Thin Sections ◽  
Staining Solution ◽  
Multiple Trials ◽  
Commercial Form

The use of lead stains for thin sections in electron microscopy is widespread. Of the various compounds, lead citrate is commonly employed. However, Reynolds' technique for its preparation from lead nitrate and sodium citrate is time-consuming. With the availability of lead citrate commercially, from at least two suppliers (K & K Laboratories, Inc., Plainview, N. Y. and Polyscience, Inc., Rydal, Pennsylvania, 19046) we have investigated the suitability of this commercial form for the staining of thin sections.We attempted to develop a technique for the simple and instantaneous preparation of a staining solution to be used fresh each time and then discarded. Following multiple trials at various concentrations, we formulated its preparation in a “cookbook” fashion involving a minimum of weighing and measuring. Previously boiled, cooled, double-distilled, millipore-filtered water, which can be stored in the refrigerator, is used. To 50 ml. of water add one pellet of sodium hydroxide (Merck) and stir until dissolved.

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