scholarly journals Use of immunogold electron microscopy and monoclonal antibodies in the identification of nuclear substructures.

1984 ◽  
Vol 32 (7) ◽  
pp. 757-765 ◽  
Author(s):  
C V Clevenger ◽  
A L Epstein
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Ultrastructural Localization ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Supramolecular Organization ◽  
Nuclear Antigens ◽  
Interchromatin Granules ◽  
Cycle Dependent ◽  
Cytochemical Technique

A cytochemical technique for the ultrastructural localization of unique nuclear antigens is reported. Using a post-embedding indirect immunogold labeling procedure, nuclear antigens in electron-dense regions of the nucleus are localized with a minimum of nonspecific staining. Using this technique and indirect immunofluorescence, a panel of antinuclear monoclonal antibodies is shown to recognize preferentially cell cycle-dependent nuclear substructures. The antigenic domains recognized include specific regions in condensed chromatin, interchromatin granules, euchromatin, and chromosomes. The specificity of antigen recognition is demonstrated with qualitative and quantitative immunogold electron microscopy and immunoblot analysis. These results reveal the existence of previously undefined supramolecular organization within the nucleus and demonstrate the utility of the immunogold procedure when monoclonal antibodies are used.

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies

1989 ◽  
Vol 93 (3) ◽  
pp. 491-500 ◽  
Author(s):  
A. Woods ◽  
T. Sherwin ◽  
R. Sasse ◽  
T.H. MacRae ◽  
A.J. Baines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Complex Mixtures ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Tubulin Isotypes ◽  
Definition Of ◽  
Attachment Zone

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

scholarly journals Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2

2020 ◽  
Vol 318 (1) ◽  
pp. F135-F147
Author(s):  
Poorichaya Somparn ◽  
Chatikorn Boonkrai ◽  
Komgrid Charngkaew ◽  
Nusara Chomanee ◽  
Kenneth G. Hodge ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Ureteral Obstruction ◽  
Collecting Duct ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Sham Operation ◽  
Autophagic Degradation ◽  
Cell Cell

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

scholarly journals Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

1992 ◽  
Vol 40 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Y Fukui ◽  
A Yamamoto ◽  
R Masaki ◽  
K Miyauchi ◽  
Y Tashiro
Keyword(s):  
Electron Microscopy ◽  
Particle Density ◽  
Protein A ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Thin Sections ◽  
Gold Particles ◽  
Rough Er

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.

scholarly journals Monoclonal Antibodies to a Subfraction of Merino Wool High-tyrosine Proteins

1986 ◽  
Vol 39 (4) ◽  
pp. 341 ◽  
Author(s):  
DR Hewish ◽  
PW French
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Immunofluorescent Staining ◽  
Immunogold Electron Microscopy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Location ◽  
Merino Wool

Monoclonal antibodies were prepared which react with members of the high-tyrosine type proteins from Merino wool. Specificity was confirmed by the use of Western transfer immunoassays and by enzyme-linked immunosorbent assay on purified fractions. Immunofluorescent staining of sections of wool follicles using the antibodies showed that the proteins were present in the developing wool shaft but that staining was asymmetric, indicating specific location of the proteins in the orthocortex of the fibres. Immunogold-electron microscopy confirmed that one of the antibodies bound to the keratin microfibril bundles.

Ultrastructural localization of Tamm—Horsfall protein in human kidney using immunogold electron microscopy

1988 ◽  
Vol 20 (3) ◽  
pp. 156-164 ◽  
Author(s):  
Robert J. Peach ◽  
William A. Day ◽  
Peter J. Ellingsen ◽  
A. Roy McGiven
Keyword(s):  
Electron Microscopy ◽  
Human Kidney ◽  
Ultrastructural Localization ◽  
Immunogold Electron Microscopy

scholarly journals The 17β-oestradiol dehydrogenase of pig endometrial cells is localized in specialized vesicles

1993 ◽  
Vol 290 (3) ◽  
pp. 777-782 ◽  
Author(s):  
J Adamski ◽  
B Husen ◽  
F Marks ◽  
P W Jungblut
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Immunogold Electron Microscopy ◽  
The Novel ◽  
Electron Dense Material ◽  
Endometrial Cells ◽  
Functional Aspects ◽  
Cytoplasmic Vesicles ◽  
Similar Appearance ◽  
High Degree

Two monoclonal antibodies against the 17 beta-oestradiol dehydrogenase of pig endometrial cells have been used in localization studies with immunogold electron microscopy. The antibodies attach both to a fraction of dehydrogenase-rich cytoplasmic vesicles isolated from homogenates and to vesicles of similar appearance in cells. The vesicles are filled with electron-dense material. Their tagging intensity indicates a high degree of specialization. Endometrial cells from mature animals contain a host of dehydrogenase vesicles, and cells from prepubertal animals only a few. Functional aspects of the novel organelle are discussed.

scholarly journals Orientation within the Exosporium and Structural Stability of the Collagen-Like Glycoprotein BclA of Bacillus anthracis

2005 ◽  
Vol 187 (15) ◽  
pp. 5310-5317 ◽  
Author(s):  
Jeremy A. Boydston ◽  
Ping Chen ◽  
Christopher T. Steichen ◽  
Charles L. Turnbough
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Triple Helix ◽  
Thermal Denaturation ◽  
Sodium Dodecyl ◽  
Basal Layer ◽  
Structural Data ◽  
Immunogold Electron Microscopy

ABSTRACT Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T m ) of 37°C, like mammalian collagen. However, rBclA trimers exhibited T m values of 84°C and 95°C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T m values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.

Ultrastructural localization of nuclear antigens during interphase in mouse 3T3 fibroblasts

1989 ◽  
Vol 67 (9) ◽  
pp. 563-574 ◽  
Author(s):  
Nathalie Chaly ◽  
Gerald St. Aubin ◽  
David L. Brown
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Ultrastructural Localization ◽  
Nuclear Pores ◽  
Thin Sections ◽  
Nuclear Ultrastructure ◽  
3T3 Fibroblasts ◽  
Nuclear Antigens ◽  
Internal Component ◽  
Immunofluorescence Labelling

Thin sections of mouse 3T3 fibroblast nuclei labelled by immunoperoxidase with anti-nuclear antibodies I1, PI1, PI2, anti-peripherin, -lamin, and -centromere have been examined in the electron microscope. Staining was compared with the corresponding immunofluorescence labelling patterns, and was correlated with nuclear ultrastructure in conventionally fixed and uranyl-lead stained samples and in unlabelled immunoperoxidase controls. Peripherin was detected at the nuclear rim in a band broader and more irregular than the lamins/lamina. The peripheral components of PI1 and PI2 appear to be localized at nuclear pores and the nuclear envelope, respectively. The internal component of PI1 staining consisted of irregular patches and strands in the nucleoplasm, closely resembling snRNP staining as reported by others. Internal PI2 labelling consisted of spots distributed apparently at random in interchromatinic regions. The spots resembled labelling by antibody I1, but were fewer and more irregular in size. Neither the PI2 nor the I1 spots were centromere associated, nor could they be correlated with specific interchromatinic structures in conventional preparations and in unlabelled controls. The results support the hypothesis that the nucleus is segregated into function-specific domains, distinguished by morphology and (or) composition from surrounding regions of the nucleus.Key words: nuclear matrix, peripherin, immunoperoxidase, electron microscopy, nuclear antigens.

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