Immunoprobe Localization by Correlative Microscopy

2000 ◽  
Vol 6 (3) ◽  
pp. 195-201
Author(s):  
Patricia G. Calarco
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Correlative Microscopy ◽  
Microtubule Organizing Center ◽  
Large Size ◽  
Organizing Center ◽  
Gamma Tubulin ◽  
High Level ◽  
And Function

Abstract Mammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

Immunoprobe Localization by Correlative Microscopy

2000 ◽  
Vol 6 (3) ◽  
pp. 195-201 ◽  
Author(s):  
Patricia G. Calarco
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Correlative Microscopy ◽  
Microtubule Organizing Center ◽  
Large Size ◽  
Organizing Center ◽  
Gamma Tubulin ◽  
High Level ◽  
And Function

AbstractMammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

scholarly journals Molecular cloning and centrosomal localization of human caltractin

1993 ◽  
Vol 90 (23) ◽  
pp. 11039-11043 ◽  
Author(s):  
V D Lee ◽  
B Huang
Keyword(s):  
Amino Acid ◽  
Calcium Binding ◽  
Animal Cell ◽  
Amino Acid Identity ◽  
Microtubule Organizing Center ◽  
Unicellular Green Alga ◽  
Organizing Center ◽  
High Level ◽  
And Function ◽  
High Degree

Caltractin, a 20-kDa calcium-binding protein, was previously purified and cloned at the DNA level from the unicellular green alga Chlamydomonas. It is a structural component of the basal body complex, the major microtubule-organizing center in Chlamydomonas and the functional homolog of the centrosome in the animal cell. Here we report the characterization of a cDNA encoding a human caltractin that shares a high degree of amino acid identity (70%) with its algal counterpart. Caltractin was identified in both HeLa and BHK cells as a 21-kDa polypeptide specifically localized to the centrosome of interphase and mitotic cells. The high level of conservation in the amino acid sequence of caltractin from algae to humans and its association with the major microtubule-organizing center in the cell suggest that caltractin plays a fundamental role in microtubule-organizing center structure and function.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

Erythrophagocytosis by Entamoeba histolytica.- Ultrastructural Study

1972 ◽  
Vol 30 ◽  
pp. 156-157 ◽  
Author(s):  
Norberto Treviño ◽  
Alfredo Feria-Velasco ◽  
I. Ruiz de Chávez
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Entamoeba Histolytica ◽  
Ultrastructural Study ◽  
Saline Solution ◽  
Physiological Saline ◽  
Ultrastructural Level ◽  
Hot Plate ◽  
Physiological Saline Solution ◽  
Axenic Conditions

Although erythrophagocytosis by various species of Entamoeba is a well known phenomenon this has not yet been studied in detail at the ultrastructural level. The present work deals with the description of the incorporation process of erythrocytes by trophozoites of E. histolytica. For this study, trophozoites of E. histolytica, HK-9:NIH strain cultured in axenic conditions and washed human erythrocytes were placed on a hot plate at 37°C in physiological saline solution. After 5 minutes, 2.5% glutarldehyde was added and the samples were processed according to conventional techniques for electron microscopy.Based upon light microscopy studies on living trophozoites in contact with erythrocytes, it seems that erythrophagocytosis only takes place in one pole of the parasite.

scholarly journals Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements

1979 ◽  
Vol 83 (3) ◽  
pp. 623-632 ◽  
Author(s):  
M Schliwa ◽  
U Euteneuer ◽  
W Herzog ◽  
K Weber
Keyword(s):  
Complete Removal ◽  
Cell System ◽  
The State ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Functional Changes ◽  
Organizing Center ◽  
Pigment Distribution ◽  
And Function ◽  
In Cells

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

The Tegument and Associated Structures of Fasciola Hepatica

1963 ◽  
Vol s3-104 (68) ◽  
pp. 505-512
Author(s):  
L. T. THREADGOLD
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Light Microscopy ◽  
Fasciola Hepatica ◽  
Golgi Bodies ◽  
New Knowledge ◽  
Pinocytotic Vesicles ◽  
The Individual ◽  
And Function ◽  
Internal Level

The cuticle of light microscopy is shown by electron microscopy to be a surface layer of protoplasm which is an extension of areas of nucleated protoplasm lying deep in the parenchyma. The cuticle therefore exists at two levels. The external level is syncytial, consisting of plateaux separated by branching valleys. This level contains apical pinocytotic vesicles, numerous mitochondria, endoplasmic membranes, large basal and other vacuoles, and dense spines. Tube-like evaginations from the base of the external level connect it to the individual areas of flask-shaped protoplasm which compose the internal level. Each of these areas of protoplasm contains a nucleus, great numbers of mitochondria, some vacuoles and diffuse inclusions, and the Golgi bodies. The histochemistry and function of the cuticle is discussed in the light of this new knowledge of cuticular ultrastructure, and a comparison is made between the cuticle of Cestoda and Trematoda.

Taxonomic application of macro and micro morphological characters of seeds in Astragalus L. (Galegeae, Fabaceae) in India

Phytotaxa ◽  
2021 ◽  
Vol 502 (2) ◽  
pp. 191-207
Author(s):  
SHIVANI KASHYAP ◽  
CHANDAN KUMAR SAHU ◽  
ROHIT KUMAR VERMA ◽  
LAL BABU CHAUDHARY
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light Microscopy ◽  
Seed Coat ◽  
Morphological Plasticity ◽  
Morphological Characters ◽  
Individual Species ◽  
Large Size ◽  
The Individual ◽  
Scanning Electron

Due to large size and enormous morphological plasticity, the taxonomy of the genus Astragalus is very complex and challenging. The identification and grouping of species chiefly based on macromorphological characters become sometimes difficult in the genus. In the present study, the micromorphology of the seeds of 30 species belonging to 14 sections of Astragalus from India has been examined applying scanning electron microscopy (SEM) along with light microscopy (LM) to evaluate their role in identification and classification. Attention was paid to colour, shape, size and surface of seeds. The overall size of the seeds ranges from 1.5–3.2 × 0.8–2.2 mm. The shape of the seeds is cordiform, deltoid, mitiform, orbicular, ovoid and reniform. The colour of seeds varies from brown to blackish-brown to black. Papillose, reticulate, ribbed, rugulate and stellate patterns were observed on the seed coat surface (spermoderm) among different species. The study reveals that the seed coat ornamentations have evolved differently among species and do not support the subgeneric and sectional divisions of the genus. However, they add an additional feature to the individual species, which may help in identification in combination with other macro-morphological features.

scholarly journals Ultrastructural Changes of the Tracheal Epithelium after Vaccination of Day-Old Chickens with the La Sota Strain of Newcastle Disease Virus

2005 ◽  
Vol 42 (5) ◽  
pp. 559-565 ◽  
Author(s):  
J. Mast ◽  
C. Nanbru ◽  
T. van den Berg ◽  
G. Meulemans
Keyword(s):  
Electron Microscopy ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Goblet Cells ◽  
Ultrastructural Level ◽  
Ultrastructural Changes ◽  
Ciliated Cells ◽  
Specific Pathogen ◽  
And Function

The progression of tracheal lesions induced by vaccination of day-old specific pathogen-free chicks with the La Sota strain of Newcastle disease virus (NDV) was examined by relating surface changes as observed by scanning electron microscopy with subcellular changes seen by transmission electron microscopy. NDV infection resulted in hypertrophy of goblet cells, their rupture, and the formation of excess mucus. Activation of goblet cells peaked within 4 days postvaccination. Afterward, the activation levels gradually decreased. At the level of the ciliated cells, a marked increase in the proportion of nonciliated to ciliated cells and later an almost complete deciliation of the tracheal surface were observed because a simple squamous to cuboidal epithelium replaced the original pseudostratified epithelium. Fifteen days postvaccination, all epithelial damage was restored. Because the observed vaccination-induced lesions are detrimental to epithelial integrity and function as a barrier against invading microorganisms, they might explain at the ultrastructural level the secondary complications of vaccination with the La Sota strain against NDV

scholarly journals Ultrastructural immunocytochemical localization of calmodulin in cultured cells.

1983 ◽  
Vol 31 (4) ◽  
pp. 445-461 ◽  
Author(s):  
M C Willingham ◽  
J Wehland ◽  
C B Klee ◽  
N D Richert ◽  
A V Rutherford ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Performic Acid ◽  
Microtubule Organizing Center ◽  
Interphase Cells ◽  
Organizing Center ◽  
Late Telophase ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
And Function ◽  
Fibroblastic Cells

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

scholarly journals Histological Variation in Plethodontid Digital Surfaces [University of Findlay]

2019 ◽  
Author(s):  
Megan Pasternak ◽  
Justin Rheubert
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Surface Morphology ◽  
Light Microscopy ◽  
Morphological Evolution ◽  
Ventral Surface ◽  
Cellular Level ◽  
Plethodontid Salamanders ◽  
And Function ◽  
Scanning Electron

Despite numerous investigations into the morphology and function of toe pads in many species, most notably anurans and geckonids, there is relatively little knowledge on salamander digit morphology. To date, toe morphology in salamanders has been limited to Desmognathus fuscus, Ambystoma maculatum, Bolitoglossa sp., and Aneides aeneus. The limited studies to date have shown variation inter- and intra-specifically but have not investigated numerous taxa within a given family which may provide deeper insights into the causes of variation (phylogenetic vs ecological pressures). Therefore, to test hypotheses concerning the presence of variation in the ventral digital surface of plethodontid salamanders, we plan to use various microscopy methodologies to view the ventral surface of the digital tips of three species from three different genera within the Plethodontidae: Desmognathus, Eurycea, and Plethodon. Toe pads will be characterized grossly using scanning electron microscopy, histologically using light microscopy, and ultrastructurally using transmission electron microscopy. Preliminary results suggest that all three species investigated display enlarged surfaces. Surface morphology (assessed via scanning electron microscopy) varies between species at a gross level concerning the shape and overall orientation of the enlarged surface. Surface morphologies include a well-developed circular pad (D. fuscus), a well-developed oval pad (P. cinereus), and a poorly developed circular pad (E. cirrigera). Furthermore, surface morphology appears to vary at the cellular level as well, with Desmognathus having polygonal squamous cells with microprojections and Eurycea having polygonal cells with nanopillars in a honeycomb arrangement. These differences may be attributed to differences in habitat preference as the three species tested include a terrestrial, semi-aquatic, and aquatic dwelling species. However, further investigation including light microscopy and enhanced scanning electron microscopy are needed. Further understanding of the morphological variation will aid in our understanding of ecomorphology and understanding of morphological evolution in amphibians.

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