scholarly journals Introduction of Tyramide Signal Amplification (TSA) to Pre-embedding Nanogold-Silver Staining at the Electron Microscopic Level

2005 ◽  
Vol 53 (2) ◽  
pp. 249-252 ◽  
Author(s):  
Seung-won Lee ◽  
Song Eun Lee ◽  
Seong Hyuk Ko ◽  
Eun Kyoung Hong ◽  
Kwang Il Nam ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Matrix Protein ◽  
Light And Electron Microscopy ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Tyramide Signal Amplification ◽  
Electron Microscopic ◽  
Simple Protocol ◽  
Tissue Antigens ◽  
A Cell

The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol.

From light to electron microscopic analysis of neurons intracellularly injected in fixed slices with “miniruby”, a fluorescent biocyttn compound

1993 ◽  
Vol 51 ◽  
pp. 290-291
Author(s):  
Wei-lin Liu ◽  
Michael T. Shipley
Keyword(s):  
Piriform Cortex ◽  
Microscopic Analysis ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Fluorescent Compound ◽  
Adult Rat ◽  
Living Slices ◽  
A Cell

Intracellular labeling of neurons in fixed slices is a very useful method for studying morphological structures of neurons both at light and electron microscopic levels. Recently, biocytin has been widely used for intracellular labeling in living slices because this molecule is highly soluble, has high electrophoretic mobility and has high affinity for avidin. However, biocytin cannot be used in fixed slices because in fixed slices membrane potential cannot be used to signify that a cell is impaled. Thus, in fixed slices it is necessary to inject cells with a fluorescent compound so that impalement and filling can be visualized under fluorescent microscope. We have developed a fluorescent biocytin compound, “Miniruby” (MR), dextran-tetramethylrhodamine-biocytin. previously, we showed that mis molecule provides excellent intracellular labels in fixed slices at the light microscopic level. Here, we demonstrate MR can also be visualized at the electron microscopic level.Fixed slices (200-400 ¼m) of adult rat olfactory bulb, piriform cortex and periaqeductal gray were used.

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 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 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 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 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.

scholarly journals Analytical electron microscopic evaluation of the effects of paraformaldehyde pretreatment on the reaction of glutaraldehyde with biogenic amines.

1982 ◽  
Vol 30 (5) ◽  
pp. 481-486 ◽  
Author(s):  
R E McClung ◽  
J Wood
Keyword(s):  
Electron Microscopy ◽  
Analytical Electron Microscopy ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Spectrographic Analysis ◽  
Electron Microscopic ◽  
X Ray ◽  
Microscopic Evaluation ◽  
Analytical Electron ◽  
Dense Product

Analytical electron microscopy was used to determine the quantitative effects of paraformaldehyde pretreatment on the formation of the biogenic amine-glutaraldehyde-chrome complex. Pretreatment with paraformaldehyde prevented the glutaraldehyde-chrome reaction with norepinephrine in the rat adrenal medulla. In contrast to the effect of paraformaldehyde on norepinephrine, pretreatment did not prevent the chrome reaction in serotonin-containing argentaffin cells of the gut. X-Ray energy spectrographic analysis revealed a significant decrease in chrome content in the paraformaldehyde treated tissue, but sufficient chrome did react to produce an electron-dense product. Thus by treating tissue with paraformaldehyde prior to the glutaraldehyde chrome procedure, serotonergic sites may be differentiated from catecholaminergic areas at the electron microscopic level.

Sign in / Sign up

Export Citation Format

Share Document