The Immunogold-Silver Staining Procedure in the Study of Freshly Suspended Langerhans Cells at the Transmission Electron Microscopic Level

A simple silver staining technique for use at the electron microscopic level, consisting only of treatment with aqueous silver nitrate at high temperature for a prolonged time, was applied to thin sections of root tip meristems of Vicia faba. This technique contrasted the fibrillar component and the granular component in interphase nucleoli as a reflection of the degree of packing. In contrast, silver impregnation was scarcely discerned in chromosomes. A comparison of silver staining and conventional double staining showed that the fibrillar centres did not always respond positively to silver. During the course from metaphase to late anaphase the nucleolus organizing secondary constriction was always seen as a heavily impregnated region and the electron density of the cytoplasm increased, probably due to dispersed nucleolar material. An argyrophilic substance began to accumulate on chromosomes in late anaphase. In the beginning of telophase a uniformly impregnated nucleolus was formed at the secondary constriction. It is concluded from these results that argyrophilic substance is associated with RNA-containing structures rather than DNA-containing structures. The silver staining technique presented here is very convenient and favourable, especially for plant cells, to detect specifically the nucleolus organizing region and to survey the nucleolar material during mitosis at the electron microscopic level.

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GLUCOSE OXIDASE AS AN ANTIGEN MARKER FOR LIGHT AND ELECTRON MICROSCOPIC STUDIES

Journal of Histochemistry & Cytochemistry ◽

10.1177/19.6.361 ◽

1971 ◽

Vol 19 (6) ◽

pp. 361-368 ◽

Cited By ~ 20

Author(s):

W. D. KUHLMANN ◽

S. AVRAMEAS

Keyword(s):

Hydrogen Peroxide ◽

Glucose Oxidase ◽

Microscopic Level ◽

Galactose Oxidase ◽

Electron Microscopic Level ◽

Staining Procedure ◽

Electron Microscopic ◽

Specific Antibodies ◽

Production Of Hydrogen ◽

Microscopic Studies

A new staining procedure for glucose oxidase at both the light and the electron microscopic level has been developed. The procedure was equally effective for the detection of galactose oxidase. It appears that the method can be applied to all oxidoreductases which catalyze the production of hydrogen peroxide. Aspergillus niger glucose oxidase and Dacytlium dendroides galactose oxidase were used to immunize rabbits and mice in order to trace the distribution of specific antibodies in the immunocytes of the popliteal lymph nodes.

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A specific ultrastructural method to reveal DNA: the NAMA-Ur.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.10.1719069 ◽

1991 ◽

Vol 39 (10) ◽

pp. 1427-1438 ◽

Cited By ~ 41

Author(s):

P S Testillano ◽

M A Sanchez-Pina ◽

A Olmedilla ◽

M A Ollacarizqueta ◽

C J Tandler ◽

...

Keyword(s):

Ultrastructural Level ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Electron Microscopic ◽

En Bloc ◽

Easy Method ◽

Transmission Electron ◽

Pre Treatment ◽

Alkali Hydrolysis

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.

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Silver Staining of Pancreatic Islets as Revealed by Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060234 ◽

1967 ◽

Vol 25 ◽

pp. 44-45

Author(s):

Kazuaki Misugi ◽

Nobuko Misugi ◽

Hiroshi Yamada

Keyword(s):

Pancreatic Islet ◽

Silver Staining ◽

Islet Cells ◽

Severe Hypoglycemia ◽

Granular Cell ◽

Electron Microscopic Level ◽

Staining Reaction ◽

Electron Microscopic ◽

Pancreatic Islet Cell ◽

D Cell

The authors had described the fine structure of a type of pancreatic islet cell, which appeared different from typical alpha and beta cells, and tentatively considered that this third type of granular cell probably represents the D cell (Figure 1).Since silver staining has been widely used to differentiate different types of pancreatic islet cells by light microscopy, an attempt to examine this staining reaction at the electron microscopic level was made.Material and Method: Surgically removed specimens from three infants who suffered from severe hypoglycemia were used. The specimens were fixed and preserved in 20% neutral formalin. Frozen sections, 30 to 40 micron thick, were prepared and they were stained by Bielschowsky's method as modified by Suzuki (2). The stained sections were examined under a microscope and islet tissues were isolated. They were fixed in 1% osmium tetroxide in phosphate buffer for one hour and embedded in Epon 812 following dehydration through a series of alcohols and propylene oxide.

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A simplified method of emulsion coating and development for quantitative electron microscopic radioautography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081188 ◽

1978 ◽

Vol 36 (2) ◽

pp. 64-65

Author(s):

K. Yoshida ◽

F. Murata ◽

S. Ohno ◽

T. Nagata

Keyword(s):

Mass Production ◽

Identical Condition ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Electron Microscopic ◽

Simplified Method ◽

Complicated Process ◽

Critical Problems ◽

Electron Microscopic Radioautography ◽

Quantitative Radioautography

IntroductionSeveral methods of mounting emulsion for radioautography at the electron microscopic level have been reported. From the viewpoint of quantitative radioautography, however, there are many critical problems in the procedure to produce radioautographs. For example, it is necessary to apply and develop emulsions in several experimental groups under an identical condition. Moreover, it is necessary to treat a lot of grids at the same time in the dark room for statistical analysis. Since the complicated process and technical difficulties in these procedures are inadequate to conduct a quantitative analysis of many radioautographs at once, many factors may bring about unexpected results. In order to improve these complicated procedures, a simplified dropping method for mass production of radioautographs under an identical condition was previously reported. However, this procedure was not completely satisfactory from the viewpoint of emulsion homogeneity. This paper reports another improved procedure employing wire loops.

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The blister of porphyria cutanea tarda: A Scanning and Transmission Electron Microscopic study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100143298 ◽

1986 ◽

Vol 44 ◽

pp. 334-335

Author(s):

Veronika Burmeister ◽

R. Swaminathan

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Basement Membrane ◽

Middle Age ◽

Porphyria Cutanea Tarda ◽

Minor Trauma ◽

Electron Microscopic ◽

Transmission Electron Microscopic Study ◽

Transmission Electron ◽

Transmission Electron Microscopic

Porphyria cutanea tarda (PCT) is a disorder of porphyrin metabolism which occurs most often during middle age. The disease is characterized by excessive production of uroporphyrin which causes photosensitivity and skin eruptions on hands and arms, due to minor trauma and exposure to sunlight. The pathology of the blister is well known, being subepidermal with epidermodermal separation, it is not always absolutely clear, whether the basal lamina is attached to the epidermis or the dermis. The purpose of our investigation was to study the attachment of the basement membrane in the blister by comparing scanning with transmission electron microscopy.

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X-Ray Microanalysis of Nickel and Cobalt Intensified Peroxidase- Antiperoxidase For Verification of Reaction Sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119168 ◽

1985 ◽

Vol 43 ◽

pp. 468-469

Author(s):

A. Angel ◽

K. Miller ◽

V. Seybold ◽

R. Kriebel

Keyword(s):

Reaction Mechanisms ◽

Visual Discrimination ◽

Osmium Tetroxide ◽

Ultrastructural Level ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Electron Microscopic ◽

X Ray ◽

Insoluble Polymer ◽

Cytochemical Reaction

Localization of specific substances at the ultrastructural level is dependent on the introduction of chemicals which will complex and impart an electron density at specific reaction sites. Peroxidase-antiperoxidase(PAP) methods have been successfully applied at the electron microscopic level. The PAP complex is localized by addition of its substrate, hydrogen peroxide and an electron donor, usually diaminobenzidine(DAB). On oxidation, DAB forms an insoluble polymer which is able to chelate with osmium tetroxide becoming electron dense. Since verification of reactivity is visual, discrimination of reaction product from osmiophillic structures may be difficult. Recently, x-ray microanalysis has been applied to examine cytochemical reaction precipitates, their distribution in tissues, and to study cytochemical reaction mechanisms. For example, immunoreactive sites labelled with gold have been ascertained by means of x-ray microanalysis.

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