scholarly journals LIGHT AND ELECTRON MICROSCOPIC LOCALIZATION OF ANTIGENS IN TISSUES EMBEDDED IN POLYETHYLENE GLYCOL WITH A PEROXIDASE-LABELED ANTIBODY METHOD

1972 ◽  
Vol 20 (12) ◽  
pp. 969-974 ◽  
Author(s):  
JOSEPH E. MAZURKIEWICZ ◽  
PAUL K. NAKANE
Keyword(s):  
Polyethylene Glycol ◽  
Anterior Pituitary ◽  
Room Temperature ◽  
Microscopic Level ◽  
Water Soluble ◽  
Electron Microscopic Level ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Antibody Method ◽  
Electron Microscopic Localization

Tissues embedded in polyethylene glycol (PEG) were used for the immunocytochemical localization of cellular antigens at the light and electron microscopic level. For demonstration of the method, growth hormone and luteinizing hormone were localized in the anterior pituitary gland of the rat embedded in PEG, using peroxidase-labeled antibodies. PEG is water-soluble, has a low melting temperature yet is firm enough to be sectioned at room temperature. Preservation of cellular ultrastructure is good. The use of PEG-embedded tissue permits precise correlation of light and electron microscopic observations of the same tissue section.

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 Electron immunocytochemical localization of enkephalin-like material in catecholamine-containing cells of the carotid body, the adrenal medulla, and in pheochromocytomas of man and other mammals.

1982 ◽  
Vol 30 (7) ◽  
pp. 682-690 ◽  
Author(s):  
I M Varndell ◽  
F J Tapia ◽  
J De Mey ◽  
R A Rush ◽  
S R Bloom ◽  
...  
Keyword(s):  
Adrenal Medulla ◽  
Carotid Body ◽  
Secretory Granules ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Antibody Technique ◽  
Dopamine Beta Hydroxylase ◽  
Carotid Bodies

Enkephalin-like immunoreactivity has been localized to electron-dense secretory granules of cat and piglet carotid bodies and adrenal medullae, horse adrenal medulla, and also to human adrenal medulla and pheochromocytomas using a gold-labeled antibody technique performed at the electron microscopic level. The same granules were also demonstrated to exhibit dopamine-beta-hydroxylase-like immunoreactivity, which suggests a granular colocalization of amines and peptides in catecholamine-storing cells.

A combined pre- and post-embedding double labeling in the central nervous system with good tissue preservation

1991 ◽  
Vol 49 ◽  
pp. 276-277
Author(s):  
A.M. Milroy ◽  
D.D. Ralston
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Highly Sensitive ◽  
The Central Nervous System

Multiple labeling at the electron microscopic level is routinely done in various parts of the central nervous system. We demonstrate that the pre-embedding tetramethylbenzidine (TMB) reaction for visualizing horseradish peroxidase (HRP) of Olucha and the slow osmication of Henry combined with a post-embedding nonetching immunogold method will also preserve good ultrastructure. Furthermore, the post-embedding immunocytochemistry of some neurotransmitters, i.e. gammaaminobutyric acid (GABA), can be done months after the tissue has been reacted for HRP and embedded in regular epon.Pre-embedding histochemistry:The use of TMB as a chromagen for the demonstration of neuronally transported HRP has both the advantage of being highly sensitive and of producing very specific needle-like crystals. Olucha et al demonstrated that one could further stabilize this reaction product with amonium heptamolybdate. Unfortunately the next step, fixation with regular osmium tetroxide, often resulted in the loss of the reaction product. However, the slow osmication with a lower pH (5.5) in the phosphate buffer at room temperature as recommended by Henry et al prevented this loss, and at the same time resulted in well preserved ultrastructure.

scholarly journals Photoconversion and electron microscopic localization of the fluorescent axon tracer fluoro-ruby (rhodamine-dextran-amine).

1993 ◽  
Vol 41 (5) ◽  
pp. 777-782 ◽  
Author(s):  
L C Schmued ◽  
L F Snavely
Keyword(s):  
Electron Microscopy ◽  
Green Light ◽  
Microscopic Level ◽  
Synaptic Connectivity ◽  
Electron Microscopic Level ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Axon Transport ◽  
Conventional Electron Microscopy ◽  
Electron Microscopic Localization

Fluoro-Ruby, the fluorescent tetramethylrhodamine-dextran-amine used to demonstrate anterograde axon transport, has been successfully photoconverted and subsequently localized by electron microscopy. The photoconversion was accomplished by irradiating the tissue with green light while bathing it in a solution containing DAB. The tissue could then be examined by brightfield microscopy or processed for conventional electron microscopy. Potential advantages of the technique include greater permanence and contrast at the light microscopic level and the ability to resolve synaptic connectivity at the 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 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 GRAMP 100: a membrane protein concentrated on secretory granule membranes and the apical cell surface in exocrine acinar cells.

1992 ◽  
Vol 40 (12) ◽  
pp. 1827-1835 ◽  
Author(s):  
S M Laurie ◽  
M B Mixon ◽  
J D Castle
Keyword(s):  
Plasma Membrane ◽  
Apical Cell ◽  
Reduced Form ◽  
Electron Microscopic Level ◽  
Apical Plasma Membrane ◽  
Cell Fractionation ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Common Component ◽  
Rat Parotid

Using a monoclonal antibody (SG10A6) raised against secretion granule membranes of the rat parotid gland, we have identified an antigen that is a common component of both exocrine pancreatic and parotid granule membranes. SG10A6 (an IgM) immunoprecipitates antigen that migrates as a single band (M(r) approximately 80 KD unreduced; M(r) approximately 100 KD reduced) and immunoblots at least two polypeptides that are similar to the reduced and nonreduced immunoprecipitated antigen. This granule-associated membrane polypeptide (GRAMP 100; named for the apparent M(r) in reduced form) is also a prominent component of plasma membrane fractions. Immunocytochemical localization at the electron microscopic level demonstrates the presence of GRAMP 100 on granule membranes, especially condensing vacuoles and exocytotic figures, and the apical plasma membrane. Lower levels of antigen are detected on basolateral plasma membrane and on peri-Golgi membranes that may be part of the endosomal system. Both the cell fractionation and immunocytochemical localization indicate that GRAMP 100 differs in distribution from GRAMP 92 and 30K SCAMPs, two other components of exocrine granule membranes identified with monoclonal antibodies. To date, no polypeptides have been identified with this approach that are exclusive components of exocrine granule membranes.

Sign in / Sign up

Export Citation Format

Share Document