scholarly journals Immunoelectron microscopic localization of estrogen receptor with monoclonal estrophilin antibodies.

1985 ◽  
Vol 33 (9) ◽  
pp. 915-924 ◽  
Author(s):  
M F Press ◽  
N A Nousek-Goebl ◽  
G L Greene
Keyword(s):  
Estrogen Receptor ◽  
Ultrastructural Localization ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Nuclear Staining ◽  
Cytoplasmic Localization ◽  
Electron Microscopic ◽  
Tissue Sections ◽  
Receptor Antibodies ◽  
Microscopic Techniques

The recent production of a series of monoclonal estrophilin (estrogen receptor) antibodies recognizing estrogen receptor derived from a wide variety of animals and target tissues permits the development of immunoelectron microscopic techniques for identifying estrogen receptor. We have determined suitable conditions for the ultrastructural localization of estrogen receptor in tissue sections. Localization of receptor was observed in the euchromatin, but not in the marginated heterochromatin or nucleoli of epithelial and stromal nuclei of human endometrium. Competition studies indicate that only estrogen receptor specifically inhibits nuclear staining. The absence of any specific cytoplasmic localization at the electron-microscopic level is consistent with earlier light-microscopic observations and suggests that the majority of the cellular pool of estrophilin exists in the nucleus of hormone-responsive cells.

scholarly journals Ultrastructural localization of guanylate cyclase in bone cells.

1991 ◽  
Vol 39 (4) ◽  
pp. 529-535 ◽  
Author(s):  
O Fukushima ◽  
C V Gay
Keyword(s):  
Plasma Membrane ◽  
Guanylate Cyclase ◽  
Bone Cells ◽  
Ultrastructural Localization ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Lead Citrate ◽  
Free Medium ◽  
Membrane Guanylate Cyclase ◽  
Electron Microscopic

Guanylyl imidodiphosphate (GMP-PNP) hydrolyzing enzyme activity as a means of detecting plasma membrane guanylate cyclase was demonstrated in osteoblasts of chicken tibial metaphysis using a lead citrate histochemical method at the electron microscopic level. Activity was not discerned in osteoclasts or osteocytes. The reaction product development was completely abolished when the sections were incubated with substrate-free or MnCl2-free medium. Guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP) was a less effective substrate than GMP-PNP, and Mn++ was a stronger stimulator than Mg++. No reaction product was observed on the plasma membrane of osteoblasts when beta-glycerophosphate or p-nitrophenylphosphate was used as substrate instead of GMP-PNP. The results implicate guanylate cyclase as a significant effector of osteoblast regulation at the site of the plasma membrane.

scholarly journals Combined use of immunoperoxidase and radioimmunocytochemistry for double immunocytochemical labeling of neurons at light and electron microscopic level.

1989 ◽  
Vol 37 (12) ◽  
pp. 1799-1809 ◽  
Author(s):  
G Alonso ◽  
P Siaud
Keyword(s):  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Double Immunostaining ◽  
Electron Microscopic ◽  
Tissue Sections ◽  
Peptide Antibody ◽  
Highly Sensitive ◽  
Antibody Complex ◽  
A New Technique ◽  
Highly Sensitive Detection

Complexes formed by binding 125I- or 3H-labeled neuropeptides to one of the two binding sites of their specific antibodies allowed specific and sensitive labeling of various peptidergic neurons, which could be detected by classical autoradiographic methods. To visualize two neuronal antigens on the same material at both light and electron microscopic level, we used a new technique of double immunocytochemical labeling, combining immunoperoxidase and radioimmunocytochemistry. The main steps of the process included: (a) indirect labeling of the first antigen by its specific antibody and by a peroxidase-labeled Fab immunoglobulin fragment directed against the primary antibody; (b) direct labeling of the second antigen by a radiolabeled peptide-antibody complex; (c) revealing of the first label in the presence of peroxidase substrate; and (d) revealing of the second label by autoradiographic treatment of tissue sections. Compared with other known techniques of double immunostaining, this technique offers major advantages for combined visualization of two neuronal antigens at the electron microscopic level: (a) two neuron types can be labeled by a pre-embedding approach, allowing highly sensitive detection of neuronal antigens throughout the 50-microns thickness of vibratome sections; (b) two primary antibodies obtained in the same species can be used to label the two antigens without any risk of crossreactions between the two successive labelings; and (c) the two labels can easily be differentiated, even when they are co-localized within the same neuron structures. Application of this double immunostaining technique is illustrated by data obtained in rat hypothalamus concerning the relationships among a variety of identified neurons and the co-localization of different neuropeptides within the same neuron system.

scholarly journals Light microscopic visualization of colloidal gold on resin-embedded tissue.

1983 ◽  
Vol 31 (12) ◽  
pp. 1394-1398 ◽  
Author(s):  
G Danscher ◽  
J O Nörgaard
Keyword(s):  
Colloidal Gold ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Metallic Silver ◽  
Frozen Sections ◽  
Electron Microscopic ◽  
Tissue Sections ◽  
Amplification Method ◽  
Glass Slides ◽  
Present Technique

RNase labeled with colloidal gold was used as a model for the present technique evolved for the light microscopic localization of gold-labeled substances in semithin resin-embedded sections. Tissue sections placed on glass slides were treated with the gold-enzyme complex and subsequently exposed to a photographic developed containing silver lactate. During the development gold particles are encapsulated in growing shells of metallic silver and gradually made visible in the light microscope. The amplification method can be applied to paraffin-embedded and frozen sections as well. This technique may prove useful as a supplement to studies utilizing colloidal gold or silver as markers normally used at the electron microscopic level.

Ultrastructural localization of soluble and insoluble 3H-methyl prednisolone as revealed by electron microscopic dry-mounting radioautography

1978 ◽  
Vol 36 (2) ◽  
pp. 40-41
Author(s):  
T. Nagata ◽  
K. Yoshida ◽  
S. Ohno ◽  
F. Murata
Keyword(s):  
Mouse Liver ◽  
Sodium Succinate ◽  
Ultrastructural Localization ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Methyl Prednisolone ◽  
Freeze Dried ◽  
Emulsion Films

IntroductionSince the recent improvements in both techniques and development in equipments at our laboratory or others, the dry-mounting procedures for radioautography facilitated the wide application of them for the study of soluble compounds such as nucleic acid precursors, amino acids, carbohydrates, lipids and steroids at both the light and electron microscopic levels. The purpose of this paper is to demonstrate ultrastructural localization of both soluble and insoluble 3H-labeled methyl prednisolone, a synthetic corticosteroid, by means of dry-mounting procedure at the electron microscopic level.Materials and MethodsMouse liver slices obtained from a male mouse and cultured HeLa cells were pulse- labeled in vitro in Eagle's MEM containing 6α-methyl prednisolone-1,2-T sodium succinate (100μCi/ml) for 1 hour.Some specimens were quickly frozen in isopentane cooled to -160°C with liquid nitrogen and freeze-dried,fixed in osmium vapour, embedded in Epon, dry-sectioned and dry-mounted according to the procedure described previously, by means of wire loops using Sakura NR-H2 emulsion containing dioctyl sodium sulfosuc- cinate in order to prevent the emulsion films from bursting while they are air dried.

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.

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