scholarly journals Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament.

1986 ◽  
Vol 34 (6) ◽  
pp. 785-793 ◽  
Author(s):  
W E Howe ◽  
F G Klier ◽  
R G Oshima
Keyword(s):  
Immunoelectron Microscopy ◽  
Intermediate Filament ◽  
Microscopic Level ◽  
Cytoskeletal Proteins ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Label ◽  
Secondary Antibodies ◽  
Endodermal Cells ◽  
20 Nm

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.

scholarly journals A New Method to Enhance Contrast of Ultrathin Cryosections for Immunoelectron Microscopy

2003 ◽  
Vol 51 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Clark L. Anderson ◽  
John M. Robinson
Keyword(s):  
Immunoelectron Microscopy ◽  
Staining Method ◽  
Microscopic Level ◽  
New Method ◽  
Electron Microscopic Level ◽  
Positive Staining ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Morphological Detail ◽  
Ultrathin Cryosections

Adequate contrast of ultrathin cryosections is crucial for evaluating morphological detail to assess immunocytochemical localization at the electron microscopic level. We have developed a positive staining method for achieving contrast in ultrathin cryosections, from tissue fixed only in paraformaldehyde, that provides excellent contrast at the electron microscopic level.

scholarly journals Biotinylphallotoxins: preparation and use as actin probes.

1989 ◽  
Vol 37 (7) ◽  
pp. 1035-1045 ◽  
Author(s):  
H Faulstich ◽  
S Zobeley ◽  
U Bentrup ◽  
B M Jockusch
Keyword(s):  
Actin Filaments ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Permeabilized Cells ◽  
High Affinity ◽  
Double Label ◽  
The One

We describe the synthesis of four phalloidin derivatives conjugated with biotin. An aminomethyldithiolane derivative of ketophalloidin was used as a reactive starter compound, and biotin residues were coupled to this molecule either directly, separated by spacer chains comprised of one or two glycyl residues, or of a 12-atom long chain constructed from succinic acid and hexamethylendiamine. Although all products still displayed a high affinity for F-actin, as seen in competition experiments with [3H]-demethylphalloidin, only the one with the longest spacer (BHPP) showed specific and high-affinity decoration of actin filaments in permeabilized cells, in conjunction with FITC-coupled avidin and fluorescence microscopy. Combined with gold-streptavidin, BHPP decorated the actin filament system at the light and electron microscopic level faithfully and with satisfactory density. Actin filaments polymerized in vitro from purified protein were not as densely labeled as had been expected. However, in all these experiments the new phalloidin probe, when combined with avidin or streptavidin, yielded clear and highly specific labeling of F-actin. Therefore, this system is useful to identify and localize actin unambiguously in microfilaments, independent of actin antibodies, and should facilitate double-label experiments on cytoskeletal components at the ultrastructural level.

scholarly journals Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy.

1987 ◽  
Vol 104 (6) ◽  
pp. 1575-1578 ◽  
Author(s):  
Y Shiomura ◽  
N Hirokawa
Keyword(s):  
Rat Brain ◽  
Immunoelectron Microscopy ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Rabbit Polyclonal Antibody ◽  
Double Label ◽  
Cross Bridges ◽  
Secondary Antibodies

Microtubule-associated protein 1A (MAP1A) and microtubule-associated protein 2 (MAP2) were shown to be colocalized on the same microtubules (MTs) within neuronal cytoskeletons by double-label immunoelectron microscopy. To investigate the electron microscopic disposition of MAP1A and MAP2 and their relationship to MTs in vivo, and to determine whether there are different subsets of MTs which specifically bind either MAP1 or MAP2, we employed a double-label immunogold procedure on rat cerebella using mouse monoclonal antibody against rat brain MAP1A and affinity-purified rabbit polyclonal antibody against rat brain MAP2. MAP1A and MAP2 were identified with secondary antibodies coupled to 10- and 5-nm gold particles, respectively. In Purkinje cell dendrites, both 10- and 5-nm gold particles were observed to be studded on the fuzzy structures attached to the same MTs. Many such structures connected MTs to each other. There was no particular MT which bound either MAP1A or MAP2 alone. Furthermore, there seemed to be no specific regions on MTs where either MAP1A or MAP2 was specifically attached. Hence, we conclude that MAP1A and MAP2 are colocalized on MTs in dendrites and assume that MAP1A and MAP2 have some interrelationship in vivo and that their interactions are responsible for forming the network of cross-bridges between MTs and MTs in neuronal cytoskeletons.

Immunoelectron Microscopy of Keratinocytes: an Improved Protocol for Flat Embedding of Cultured Cells Using Lowicryl K4M

2000 ◽  
Vol 6 (S2) ◽  
pp. 334-335
Author(s):  
C. Bauer ◽  
V. Vasioukhin ◽  
M. Yin ◽  
E. Fuchs
Keyword(s):  
Immunoelectron Microscopy ◽  
Cell Cultures ◽  
Cultured Cells ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Additional Information ◽  
Culture Models ◽  
Lowicryl K4m ◽  
Immunohistochemical Studies

In cell culture models various and distinct manipulations can be performed in a defined environment. This makes cell cultures very popular for immunohistochemical studies. Such studies are mostly performed at the light microscopic level where fluorescent probes and confocal microscopy provide detailed insights into the distribution and localization of antigens inside cells. In many such cases further studies at the electron microscopic level would give additional information and an often more detailed view. As compared to light microscopy not only is the resolution much higher, but also and even more importantly labeling information is completed with a wealth of cytological details.This said it is surprising that studies on cell cultures using immunoelectron microscopy are rarely seen. Even more rare are reports in which monolayer cells are fixed and processed in situ without prior trypsinization. Such studies are essential though when cytoarchitecture is of importance or especially when cell-cell adhesion is an issue.

scholarly journals The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.

1992 ◽  
Vol 40 (6) ◽  
pp. 883-888 ◽  
Author(s):  
H Shimizu ◽  
A Ishida-Yamamoto ◽  
R A Eady
Keyword(s):  
Basement Membrane ◽  
Normal Skin ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Specificity And Sensitivity ◽  
Structural Preservation ◽  
Secondary Antibodies ◽  
High Degree

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.

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

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.

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

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.

scholarly journals FIXATION OF NEURAL TISSUES FOR ELECTRON MICROSCOPY BY PERFUSION WITH SOLUTIONS OF OSMIUM TETROXIDE

1962 ◽  
Vol 12 (2) ◽  
pp. 385-410 ◽  
Author(s):  
Sanford L. Palay ◽  
S. M. McGee-Russell ◽  
Spencer Gordon ◽  
Mary A. Grillo
Keyword(s):  
Electron Microscopy ◽  
Nervous System ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Neural Tissues ◽  
Structural Relations ◽  
Cellular Components

This paper describes in detail a method for obtaining nearly uniform fixation of the nervous system by vascular perfusion with solutions of osmium tetroxide. Criteria are given for evaluating the degree of success achieved in the preservation of all the cellular components of the nervous system. The method permits analysis of the structural relations between cells at the electron microscopic level to an extent that has not been possible heretofore.

scholarly journals Lymphocyte Lysosomes and Lysosomal Enzymes in Chronic Lymphocytic Leukemia

Blood ◽  
1973 ◽  
Vol 41 (4) ◽  
pp. 511-518 ◽  
Author(s):  
Steven D. Douglas ◽  
Georg Cohnen ◽  
Erika KÖnig ◽  
GÜnter Brittinger
Keyword(s):  
Acid Phosphatase ◽  
Lysosomal Enzymes ◽  
Microscopic Level ◽  
Lymphocytic Leukemia ◽  
Electron Microscopic Level ◽  
Acid Hydrolase ◽  
Electron Microscopic ◽  
Normal Cells ◽  
Biochemical Studies ◽  
Cell Profile

Abstract Electron microscopic cytochemical and biochemical studies of lysosomal markers have been performed in unstimulated normal and chronic lymphotic leukemia (CLL) lymphocytes. Decreased activities of the lysosomal enzymes acid phosphatase and β-glucuronidase but not of the nonlysosomal enzyme malate dehydrogenase were observed in CLL lymphocytes as compared to normal cells. At the electron microscopic level, the number of membrane-bounded acid phosphatase-positive organelles was diminished in CLL cells. (Average 1.07 per cell profile in normal cells and 0.17 in CLL lymphocytes). The findings indicate that the diminution of acid hydrolase activities in CLL lymphocytes is most likely due to a reduced number of lysosomes, rather than to a diminished enzyme content of these organelles.

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