scholarly journals A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies.

1993 ◽  
Vol 41 (3) ◽  
pp. 447-454 ◽  
Author(s):  
J Peränen ◽  
M Rikkonen ◽  
L Kääriäinen
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Guanidine Hydrochloride ◽  
Specific Antibodies ◽  
Permeabilized Cells ◽  
Immunofluorescence Localization ◽  
Antigenic Sites ◽  
Cell Architecture ◽  
Simple Protocol ◽  
Triton X

We describe a simple protocol that allows the retrieval of masked or hidden intracellular antigens in cultured cells. The protocol is based on the exposure of paraformaldehyde-fixed and Triton X-100-permeabilized cells to guanidine hydrochloride (GdnHCl). We used it for localization of different antigens in BHK-21 cells by immunofluorescence microscopy. Our results showed that five out of six antibodies, initially unreactive, became excellent localization tools when used in conjunction with GdnHCl. Denaturation of fixed cells with GdnHCl did not affect the overall cell architecture, when monitored with different organelle-specific antibodies. In two cases out of 17 the antigenic sites were lost after denaturation. However, this problem could be partially overcome by including low amounts of glutaraldehyde in the fixative. We think that this method could be generally useful in immunofluorescence localization studies, particularly in cases where the antibodies are known to react only with denatured antigens.

Simple method for extracting RNA from cultured cells and tissue with guanidine salts

1993 ◽  
Vol 39 (7) ◽  
pp. 1408-1411 ◽  
Author(s):  
R Zolfaghari ◽  
X Chen ◽  
E A Fisher
Keyword(s):  
Cultured Cells ◽  
Guanidine Hydrochloride ◽  
Placental Tissue ◽  
Animal Origin ◽  
Clinical Samples ◽  
Simple Method ◽  
Simple Protocol ◽  
Human Placental Tissue ◽  
Polymerase Chain

Abstract We have developed a simple protocol for isolating RNA from both cell culture and tissue from human and animal sources, using guanidine thiocyanate and guanidine hydrochloride, but no organic solvents. The protocol reproducibly yielded 15 to 25 micrograms of high-quality RNA per 10(6) cells of human and animal origin and 1 to 1.1 mg of RNA per gram of human placental tissue. The RNA so obtained was ribonuclease-free and not contaminated by DNA. It was suitable for reverse transcription-polymerase chain reaction, Northern blot analysis, and in vitro expression of proteins. Thus, the molecular assessment of both research and clinical samples can be readily and reliably initiated by the application of this protocol.

Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy

1992 ◽  
Vol 101 (4) ◽  
pp. 731-743 ◽  
Author(s):  
M.A. Melan ◽  
G. Sluder
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Protein S ◽  
Soluble Proteins ◽  
Differential Extraction ◽  
Preferential Localization ◽  
Net Charges ◽  
Triton X

Immunofluorescence microscopy is widely used to characterize the cellular distribution of both soluble and structural proteins. Control experiments generally address only the specificity of the antibodies used. The permeabilization/fixation conditions used to prepare cells for antibody application are assumed to preserve faithfully the in vivo distributions of the protein(s) being examined. We systematically tested the extent to which soluble proteins are redistributed into inappropriate locations and are differentially extracted from native locations during the permeabilization and fixation of the cells before antibody application. We separately introduce six soluble FITC-conjugated proteins of different net charges and sizes into living cultured cells. The labeled proteins do not adhere to the external surfaces of living cells and are evenly distributed throughout the cytoplasm with the larger proteins being excluded from the nucleus. The cells are then prepared as if for immunofluorescence using several conditions that encompass many of the methods commonly used for this purpose. Cells permeabilized with 0.1-0.2% Triton X-100 before fixation with 3.7% paraformaldehyde show a striking localization of all but one of the test proteins to the nucleus and/or nucleoli of 60–80% of labeled cells. Punctate cytoplasmic labeling and cytoskeletal-like arrays of labeled protein are also observed. Extraction with 1% detergent prior to fixation removes most but not always all of the exogenous proteins from the cell remnants. Permeabilization of cells with 0.1% detergent after paraformaldehyde fixation leaves a reticular, uneven cytoplasmic distribution of the labeled proteins, and some of the larger proteins are redistributed to the nuclei. Direct fixation/permeabilization with -20 degrees C methanol largely preserves the in vivo distributions of fluorescent proteins with some preferential localization of these proteins to nuclei, nucleoli and the perinuclear region. These results show that misleading apparent localizations of soluble proteins can result from their redistribution and/or differential extraction during the preparation of cells for primary antibody application.

Efficient Biginelli Synthesis of 2-Aminodihydropyrimidines under Microwave Irradiation

Synlett ◽  
2018 ◽  
Vol 29 (08) ◽  
pp. 1047-1054 ◽  
Author(s):  
Fulvia Felluga ◽  
Fabio Benedetti ◽  
Federico Berti ◽  
Sara Drioli ◽  
Giorgia Regini
Keyword(s):  
Microwave Irradiation ◽  
Guanidine Hydrochloride ◽  
Reaction Times ◽  
Conventional Heating ◽  
Dicarbonyl Compounds ◽  
Simple Protocol ◽  
Twofold Excess ◽  
General Method

A practical and general method for the Biginelli cyclocondensation of guanidine with aldehydes and β-dicarbonyl compounds is described and illustrated with the synthesis of a set of 26 functionalized 2-amino-3,4-dihydropyrimidines. The simple protocol involves the ­microwave-mediated reaction of a twofold excess of guanidine hydrochloride with the required reaction partners in an alcohol at 120 °C. Yields are generally good, with short reaction times and a simple workup. The scope is considerably wider than that of similar reactions ­carried out under conventional heating.

scholarly journals Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

1978 ◽  
Vol 77 (3) ◽  
pp. R27 ◽  
Author(s):  
M Osborn ◽  
RE Webster ◽  
K Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscope ◽  
Immunofluorescence Microscopy ◽  
Uranyl Acetate ◽  
Tubulin Antibodies ◽  
Ptk2 Cell ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Microtubular Structures

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

scholarly journals Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2442-2451
Author(s):  
M Molino ◽  
M Di Lallo ◽  
N Martelli ◽  
G de Gaetano ◽  
C Cerletti
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Fluorescein Isothiocyanate ◽  
Adenosine Diphosphate ◽  
Cathepsin G ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Antigenic Sites ◽  
Triton X ◽  
Von Willebrand ◽  
Willebrand Factor

Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC- 1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA- fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb- IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.

Properties of microtubule-free cortical residues isolated from Paramecium tetraurelia

1989 ◽  
Vol 92 (3) ◽  
pp. 427-432 ◽  
Author(s):  
N.E. Williams ◽  
J.E. Honts ◽  
K.R. Stuart
Keyword(s):  
Immunofluorescence Microscopy ◽  
High Ionic Strength ◽  
Evolutionary Relationships ◽  
Antigenic Relationship ◽  
Paramecium Tetraurelia ◽  
Structure Development ◽  
Shape Preserving ◽  
Alpha Spectrin ◽  
Triton X ◽  
Cortical Architecture

We have found that shape-preserving residues devoid of microtubules can be prepared from Paramecium using Triton X-100 at high ionic strength. These residues contain many proteins, including one showing antigenic relationship to chicken alpha-spectrin, and three showing antigenic relationship to Tetrahymena cortical proteins. These antigens have been localized by immunofluorescence microscopy, and the isolated cortical residues have been characterized ultrastructurally. These preparations should be useful in detailed studies of the structure, development and evolutionary relationships of cortical architecture in ciliated protozoa.

Extracellular ciliary axonemes associated with the surface of smooth muscle cells of ctenophores

1989 ◽  
Vol 94 (4) ◽  
pp. 713-724
Author(s):  
S. Tamm ◽  
S.L. Tamm
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Direct Contact ◽  
Immunofluorescence Microscopy ◽  
Surface Membrane ◽  
Muscle Cells ◽  
Basal Bodies ◽  
Unique Role ◽  
Antigenic Sites ◽  
Smooth Muscle Function

We describe the first example of bare ciliary axonemes existing outside eukaryotic cells. The axonemes run in longitudinal invaginations of the surface membrane of giant smooth muscle cells in ctenophores. No motility of the surface-associated axonemes has been detected in living muscles. The axonemes are truly extracellular and in direct contact with the extracellular matrix (mesoglea), as shown by the ultrastructural tracer horseradish peroxidase. The axonemes appear partially degraded and disorganized, and individual doublet microtubules are difficult to distinguish. Nevertheless, immunofluorescence microscopy shows that the axonemes retain antigenic sites reacting with mouse monoclonal anti-beta-tubulin. The origin of the extracellular axonemes is unknown: no attached basal bodies (extracellular or intracellular) have been found. The muscle-associated axonemes may play a unique role in smooth muscle function and/or development, and may be related to the evolution of muscle cells in soft-bodied invertebrates that exploit cilia for a wide variety of functions.

Direct visualization of the 10-nm (100-A)-filament network in whole and enucleated cultured cells

1978 ◽  
Vol 31 (1) ◽  
pp. 393-409
Author(s):  
J.V. Small ◽  
J.E. Celis
Keyword(s):  
Cytochalasin B ◽  
Cultured Cells ◽  
Marked Effect ◽  
High Electron ◽  
Perinuclear Region ◽  
Structural Elements ◽  
3T3 Cells ◽  
10 Nm Filaments ◽  
Triton X ◽  
Filament Network

Following extraction of actomyosin and tubulin from cultured cells treated with Triton X-100, a cytoskeleton remains which is composed predominantly of the cell nucleus encompassed by a network of 10-nm filaments. After negative staining the dense perinuclear region appears as a densely woven filament net punctuated by patches of high electron density. Enucleation of 3T3 cells with cytochalasin B gives rise to karyoplasts surronunded by 10-nm filaments and cytoplasts in which 10-nm filaments remain situated in the central region of the cytoplasm. While the 10-nm filaments occurred mainly as single filaments in human skin fibroblasts and 3T3 cells, in epithelioid PtK1 and PtK2 cells they were commonly associated in prominent meandering bundles. In addition, in these latter cells after Triton extraction the remaining ribosomes were bound specifically to the 10-nm-filament net. After exposure of 3T3 cells to cytochalasin B the 10-nm filaments formed branches that radiated from the perinuclear region into the immobile cell extensions. Concavalin A had no marked effect on the distribution of the 10-nm-filament net. The results suggest that the 10-nm filaments act primarily as structural elements, serving, in particular, to support and constrain the nucleus in its position in the cell.

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