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 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.

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.

X-Ray Analysis of Cytochemical Reaction Products in Synaptic Areas

1976 ◽  
Vol 34 ◽  
pp. 6-7 ◽  
Author(s):  
J. Wood
Keyword(s):  
Electron Microscopic Level ◽  
Junctional Complex ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
X Ray ◽  
The Past ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Cytochemical Reaction

Specific cytochemical reactions have been instrumental in the illucidation of compounds within tissues, whether these compounds are hormones, enzymes, or molecules, such as certain nerve transmitter agents. Many cytochemical reaction products depend upon some complex, which is an electron dense deposit. Several types of cytochemical procedures can be used to visualize agents related to synaptic transmission at the junctional complex. One method which has been used with considerable success has been the cytochemical localization of biogenic amines (BAs), i.e., norepinephrine (NE) and dopamine (DA). For the past few years, a chrome complex formed with certain BAs and glutaraldehyde has been utilized to localize BAs at the electron microscopic level and the specificity of the reaction has been verified biochemically.

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 Catecholamine innervation of enkephalinergic neurons in guinea pig hypothalamus: demonstration by an in vitro autoradiographic technique combined with a post-embedding immunogold method.

1988 ◽  
Vol 36 (5) ◽  
pp. 533-542 ◽  
Author(s):  
V Mitchell ◽  
J C Beauvillain ◽  
P Poulain ◽  
M Mazzuca
Keyword(s):  
Guinea Pig ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Morphological Evidence ◽  
Osmic Acid ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Thin Sections

To study the relationship between the catecholamine (CA) nerve endings and the enkephalinergic cell bodies in the magnocellular dorsal nucleus (MDN) of guinea pig hypothalamus, double-labeling experiments were performed on the same tissue section at the electron microscopic level. An in vitro autoradiographic (ARG) method for [3H]-norepinephrine (NE) or [3H]-dopamine (DA) was combined with a post-embedding immunogold cytochemical technique for Met-enkephalin (Met-enk) in colchicine-treated animals. Hypothalamic slices (450 micrograms) were perfused with [3H]-NE or [3H]-DA at the fluid-gas interface, then fixed by immersion with glutaraldehyde and osmic acid. Semi-thin sections processed from the thickness of the slices showed adequate penetration of the tracers to all parts of the tissue. Frontal sections permitted visualization of some CA-uptake structures distributed around the cells. At the ultrastructural level, preservation appeared good on about 60% of the thickness of slices, and [3H]-CA structures were easily distinguished. Ultra-thin sections were successively incubated with Met-enk and colloidal gold-labeled antisera, followed by ARG processing. At the electron microscopic level, the good integrity of the tissue made possible visualization of [3H]-CA nerve terminals making synaptic contacts with enkephalinergic perikarya. These results provide morphological evidence for direct catecholaminergic control of enkephalinergic neurons of the MDN.

scholarly journals A specific ultrastructural method to reveal DNA: the NAMA-Ur.

1991 ◽  
Vol 39 (10) ◽  
pp. 1427-1438 ◽  
Author(s):  
P S Testillano ◽  
M A Sanchez-Pina ◽  
A Olmedilla ◽  
M A Ollacarizqueta ◽  
C J Tandler ◽  
...  
Keyword(s):  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
En Bloc ◽  
Easy Method ◽  
Transmission Electron ◽  
Pre Treatment ◽  
Alkali Hydrolysis

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.

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

1980 ◽  
Vol 28 (12) ◽  
pp. 1312-1318 ◽  
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.

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.

A New Technique for the Light and Electron Microscopic Study of Individual Cerebro-Spinal Fluid Cells in a Mona Plane Layer

1976 ◽  
Vol 34 ◽  
pp. 362-363
Author(s):  
L.A. Dell
Keyword(s):  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Plane Layer ◽  
Ultrastructural Level ◽  
Spinal Fluid ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Easy Method ◽  
Cell Pellet ◽  
A New Technique

A new method has been developed which readily offers the microscopist a possibility for both light and electron microscopic study of selected cells from the cerebrospinal fluid. Previous attempts to examine these cells in the spinal fluid at the ultrastructural level were based on modifications of cell pellet techniques developed for peripheral blood. These earlier methods were limited in application by the number of cells in spinal fluid required to obtain a sufficient size pellet and by the lack of an easy method of cellular identification between the light and electron microscopic level. The newly developed method routinely employs microscope slides coated with Siliclad and tungsten oxide for duplicate cytocentrifuge preparations of diagnostic spinal fluid specimens. Work done by Kushida and Suzuki provided a basis for our use of the metal oxide.

Sign in / Sign up

Export Citation Format

Share Document