"Immunocytochemistry in the vestibular endorgans: analysis of light microscopic level to electron microscopic level."

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.5.7682231 ◽

1993 ◽

Vol 41 (5) ◽

pp. 777-782 ◽

Cited By ~ 23

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.

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Immunocytochemical localization of renin in the submandibular gland of the mouse.

Journal of Histochemistry & Cytochemistry ◽

10.1177/26.10.363930 ◽

1978 ◽

Vol 26 (10) ◽

pp. 855-861 ◽

Cited By ~ 24

Author(s):

E Gresik ◽

A Michelakis ◽

T Barka ◽

T Ross

Keyword(s):

Submandibular Gland ◽

Adult Mouse ◽

Secretory Granules ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Light Microscopic Level ◽

Electron Microscopic ◽

Granular Convoluted Tubule ◽

Enzyme Method ◽

Unlabeled Antibody

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.

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Immunocytochemical localization of calmodulin and a heat-labile calmodulin-binding protein (CaM-BP80) in basal ganglia of mouse brain.

The Journal of Cell Biology ◽

10.1083/jcb.84.1.66 ◽

1980 ◽

Vol 84 (1) ◽

pp. 66-76 ◽

Cited By ~ 194

Author(s):

J G Wood ◽

R W Wallace ◽

J N Whitaker ◽

W Y Cheung

Keyword(s):

Binding Protein ◽

Brain Area ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Light Microscopic Level ◽

Neuronal Somata ◽

Electron Microscopic ◽

Calmodulin Binding ◽

Heat Labile ◽

Dividing Cells

Antisera to calmodulin, a Ca2%-dependent modulator protein, and a heat-labile calmodulin-binding protein have been used to localize these proteins in mouse caudate-putamen. The two proteins appear to be located at identical sites in this brain area. At the light microscopic level, calmodulin and calmodulin-binding protein are found within the cytoplasm and processes of large cells. At the electron microscopic level the proteins are associated with neuronal elements only, primarily at postsynaptic sites within neuronal somata and dendrites. Within the dendrites the immunocytochemical label is associated predominantly with the postsynaptic density and dendritic microtubules. These results are in accord with recent biochemical and immunihistochemical studies of calmodulin in brain and in dividing cells. Thus, calmodulin and the heat-labile calmodulin-binding protein may play a role in the nervous system at the site of neurotransmitter action and at the level of microtubular function.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147296 ◽

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.

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Immunocytologic analyses of 10 nm intermediate filaments in the nervous system of Myxicola.

Journal of Histochemistry & Cytochemistry ◽

10.1177/28.12.7014711 ◽

1980 ◽

Vol 28 (12) ◽

pp. 1312-1318 ◽

Cited By ~ 8

Author(s):

L F Eng ◽

R J Lasek ◽

J W Bigbee ◽

D L Eng

Keyword(s):

Nervous System ◽

Glial Cells ◽

Staining Pattern ◽

Ultrastructural Level ◽

Microscopic Level ◽

Electron Microscopic Level ◽

Antigenic Determinants ◽

Light Microscopic Level ◽

Neurofilament Protein ◽

Electron Microscopic

Antibodies prepared in rabbits against Myxicola infundibulum neurofilaments have been employed to stain neurofilaments immunohistochemically in intact Myxicola infundibulum nervous tissue. Paraffin-embedded and frozen sections (5--6 mu) were examined at the light microscopic level with Sternberger's peroxidase-antiperoxidase method, and Vibratome (20--40 mu) sections were studied at the ultrastructural level with Nakane's conjugated peroxidase method. The neurofilament antibody stained only neurons and axons at the light microscopic level. The staining pattern at the electron microscopic level corresponded to the neurofilaments within axons and neurons. Glial cells, which surround the axons, contain large bundles of filaments that resemble astrocytic filaments in mammalian astrocytes. These filaments do not stain with the anti-neurofilament antibody. Neurons, neurofilaments, glial cells, glial filaments, and nonnervous tissue showed no peroxidase staining when specific antiserum absorbed with neurofilaments was used. These structures were also unstained when antiserum to the glial fibrillary acidic protein of mammalian central nervous system astrocytes was substituted for the neurofilament antiserum. Therefore, in Myxicola infundibulum, the antigenic determinants of the neurofilament protein, as recognized immunohistochemically by anti-neurofilament protein antibodies, are not shared with those of glial filaments.

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.6.2422254 ◽

1986 ◽

Vol 34 (6) ◽

pp. 785-793 ◽

Cited By ~ 9

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.12.2.385 ◽

1962 ◽

Vol 12 (2) ◽

pp. 385-410 ◽

Cited By ~ 393

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.

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