Detection of Cytoskeletal Proteins in Cultured Cells at the Ultrastructural Level

Langanger, G.; Mey, J. De

doi:10.1016/b978-0-12-140407-9.50007-6

Cited by 6 publications

(3 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The staining of F-actin and vinculin was performed according to the method of Clubb and Shivers (1996), with some modifications. After the end of the culture period, Sertoli cells grown on non-fluorescence glass chamber slides (Nunc, USA) were briefly washed with cytoskeleton-stabilizing buffer (CSB) containing 137 mM NaCl, 5 mM KCl, 1.1 mM Na 2 HPO 4 , 0.4 mM KH 2 PO 4 , 2 mM MgCl 2 , 2 mM EGTA, 5 mM PIPES, 5.5 mM glucose, pH 6.1 (Langanger, 1989) before being fixed with 3.7% paraformaldehyde in cytoskeletonstabilizing buffer, for 20 min at room temperature. Once fixed, the cells were incubated with 0.1% glycine in CSB for 5 min, permeabilized with 0.5% Triton X-100 in CSB for 3 min and washed extensively with CSB.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

The effect of 12‐O‐tetradecanoylphorbol‐13‐acetate on Sertoli cell morphology and cytoskeletal organization: an in vitro study by means of cryo‐SEM and fluorescent techniques

Panagopoulou

Kouloukoussa

Voloudaki-Baltatzi

et al. 2002

Biology of the Cell

View full text Add to dashboard Cite

By means of cryo-scanning electron microscopy (cryo-SEM) and fluorescent techniques, evidence is provided on how 12-O-tetradecanoylphorbol-13-acetate (TPA) affects Sertoli cell morphology and F-actin and vinculin organization in vitro. In order to visualize the morphological changes, the cells were observed with cryo-SEM. F-actin was localized using rhodamine (TRI)-phalloidin and vinculin using a primary monoclonal antibody and a second TRI-conjugated antibody. The results indicate that after the addition of 10(-7) M TPA, Sertoli cells begin to round up and their cytoplasm is retracted towards a central region. Actin bundle organization is disrupted and vinculin assumes a punctuate distribution throughout the cell. Thus, the reorganization of actin and vinculin and subsequent changes in cell morphology seem to be brought about by TPA affecting not only actin but also the protein vinculin which interacts with actin. A discussion is made concerning the effect of TPA on cytoskeletal reorganization, which is closely related to cell transformation.

show abstract

Section: Fluorescence Microscopymentioning

confidence: 99%

The effect of 12‐O‐tetradecanoylphorbol‐13‐acetate on Sertoli cell morphology and cytoskeletal organization: an in vitro study by means of cryo‐SEM and fluorescent techniques

Panagopoulou

Kouloukoussa

Voloudaki-Baltatzi

et al. 2002

Biology of the Cell

View full text Add to dashboard Cite

show abstract

“…Intracellular antigens may also be localized if the cells can be permeabilized by treatment with detergents. The demonstration of cytoskeletal elements in cultured cells is a strikingly visual example of this technique (Langaner & De Mey, 1989), but one should interpret data obtained by this technique with some caution. It is essential that the antigen of interest is anchored well enough to avoid partial or total extraction or translocation during the permeabilization process.…”

Section: Isolated Cells or Monolayersmentioning

confidence: 99%

Immunocytochemical strategies for electron microscopy: choice or compromise

Skepper

2000

Journal of Microscopy

View full text Add to dashboard Cite

SummaryThis review attempts to describe the major immunocytochemical strategies that are in current use for transmission electron microscopy. Emphasis is placed on suggesting the most appropriate application of each technique that is described. The review is not intended to identify the microscopist's`cure all' technique for immunocytochemistry but rather to describe what techniques are available and what are their relative merits. Expensive equipment and technically complicated methods are not always necessary to collect robust data. I hope to present a review that will enable its readers to select the most profitable technique (or compromise) available, to reflect both their resources and their ability to address their biological questions with vigour.

show abstract

“…Oocytes were treated following the procedure adapted from the method described by Langanger and De Mey (1989) to have a good preservation of the cytoskeletal structures. Oocytes were rinsed 1 sec in PHEM buffer (60 mM PIPES, 25 mM HEPES, 2 mM MgCl,, and 10 mM EGTA), extracted for 5 min in PHEM containing 0.2% Triton X-100, before a 2 min prefixation with 0.3% glutaraldehyde in PHEM containing 0.5% Triton X-100.…”

Section: Electron Microscopymentioning

confidence: 99%

Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: A study by confocal laser scanning microscopy

Zernicka‐Goetz

Kubiak

Antony

et al. 1993

Molecular Reproduction Devel

View full text Add to dashboard Cite

In metaphase II arrested rat oocytes (M II), microtubules were found in the taper-shaped meiotic spindle and in the cytoplasm as asters and free microtubules. Whereas spindle microtubules were acetylated, those located in the cytoplasm were not. Cytoplasmic microtubules were also labile as assessed by mild cooling. In contrast to mouse oocytes, rat microtubule organizing centers (MTOCs) did not react with MPM-2 antibody by immunofluorescence despite the fact that this antibody reacts with several proteins as shown by immunoblot. However, cytoplasmic MTOCs in M II-arrested rat oocytes could be detected by their nucleating capacity in the presence of taxol, a drug that induced the formation of numerous cytoplasmic asters. In addition, taxol caused a change in the spindle shape and the formation of astral microtubules at the spindle poles. Meiotic spindles (as well as chromosomes devoid of microtubules after nocodazole-treatment) were overlaid by an actin-rich domain. Spontaneous abortive activation led to the extrusion of the second polar body followed by another metaphase arrest--metaphase III; however, normal spindles did not form and dispersed chromosomes surrounded by microtubules were observed. Electron microscopic studies confirmed these observations and revealed that the kinetochores, are located deep within the chromosomes in contrast to mouse kinetochores, and this might be responsible for the absence of a metaphase III spindle in the rat oocyte. Induced activation caused transition to interphase with the formation of a characteristic microtubule network. This study shows that there are several significant differences in the cytoskeletal organization of rat and mouse oocytes.

show abstract

Detection of Cytoskeletal Proteins in Cultured Cells at the Ultrastructural Level

Cited by 6 publications

References 28 publications

The effect of 12‐O‐tetradecanoylphorbol‐13‐acetate on Sertoli cell morphology and cytoskeletal organization: an in vitro study by means of cryo‐SEM and fluorescent techniques

The effect of 12‐O‐tetradecanoylphorbol‐13‐acetate on Sertoli cell morphology and cytoskeletal organization: an in vitro study by means of cryo‐SEM and fluorescent techniques

Immunocytochemical strategies for electron microscopy: choice or compromise

Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: A study by confocal laser scanning microscopy

Contact Info

Product

Resources

About