Immuno-EM Localization of GFP-tagged Yolk Proteins in<i>C. Elegans</i>Using Microwave Fixation

Paupard, Marie-Christine; Miller, A.; Grant, Barth D.; Hirsh, David; Hall, David H.

doi:10.1177/002215540104900803

Cited by 46 publications

(30 citation statements)

References 21 publications

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“…For simplicity, we hoped to use direct visualization of GFP fluorescence to locate the expressing cells in semithin sections. Although there are many examples of immunogold localization of GFP in the literature (Shiao et al 2000;Paupard et al 2001;Ward and Moss 2001;Thomsen et al 2002), and at least one report of immunofluorescence localization in sections of LR White-embedded material (Herken et al 1988), we found no mention of whether GFP fluorescence could be visualized directly in LR White-embedded material or whether the fluorescence and the antigenicity of GFP would be retained after overnight fixation in low concentrations of glutaraldehyde. Although GFP fluorescence previously was reported to survive glutaraldehyde fixation, the length of fixation and the concentration of fixative were not given (Chalfie et al 1994).…”

contrasting

confidence: 62%

Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

Luby-Phelps

Ning

Fogerty

et al. 2003

J Histochem Cytochem.

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S U M M A R YWe have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1-m sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.

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contrasting

confidence: 62%

Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

Luby-Phelps

Ning

Fogerty

et al. 2003

J Histochem Cytochem.

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“…Serial thin sections were collected on slot grids and post-stained with uranyl acetate and lead citrate, and examined with a Philips CM10 electron microscope. To fix young L1s from RNAi experiments, animals were exposed to microwave irradiation during the primary fixation in buffered aldehydes, using a model 3450 oven (Ted Pella) at half power (Paupard et al, 2001). Subsequent fixation steps follow our normal protocols (Hall, 1995).…”

Section: Electron Microscopymentioning

confidence: 99%

Laminin α subunits and their role inC. elegansdevelopment

et al. 2003

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Laminins are heterotrimeric (α/β/γ) glycoproteins that form a major polymer within basement membranes. Different α, β and γ subunits can assemble into various laminin isoforms that have different, but often overlapping, distributions and functions. In this study, we examine the contributions of the laminin α subunits to the development of C. elegans. There are two α, one β and one γ laminin subunit, suggesting two laminin isoforms that differ by their α subunit assemble in C. elegans. We find that near the end of gastrulation and before other basement membrane components are detected, the α subunits are secreted between primary tissue layers and become distributed in different patterns to the surfaces of cells. Mutations in either α subunit gene cause missing or disrupted extracellular matrix where the protein normally localizes. Cell-cell adhesions are abnormal: in some cases essential cell-cell adhesions are lacking, while in other cases, cells inappropriately adhere to and invade neighboring tissues. Using electron microscopy, we observe adhesion complexes at improper cell surfaces and disoriented cytoskeletal filaments. Cells throughout the animal show defective differentiation, proliferation or migration, suggesting a general disruption of cell-cell signaling. The results suggest a receptor-mediated process localizes each secreted laminin to exposed cell surfaces and that laminin is crucial for organizing extracellular matrix, receptor and intracellular proteins at those surfaces. We propose this supramolecular architecture regulates adhesions and signaling between adjacent tissues.

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“…Some recent papers have indicated preferential immunolabeling density by MWI (Rangell & Keller 2000, Paupard et al 2001. These results were obtained using acrylic resin (such as LR Gold, LR White) or frozen sections.…”

Section: Effect Of Mwi On Fixation and Immunolabeling Of Plastic-embementioning

confidence: 96%

“…The MWI has been widely utilized to advance biological processing: tissue fixation, histochemistry, antigen retrieval, and in situ hybridization (Shi et al 1991, Login et al 1995, Van Ginneken et al 1998, Rangell & Keller 2000, Mitchell et al 2001, Paupard et al 2001. The great increase in molecular motion induced by MWI enables fixatives to soak rapidly and deeply into cells and tissues (Boon et al 1990, Rangell & Keller 2000.…”

Section: Effect Of Mwi On Fixation and Immunolabeling Of Plastic-embementioning

confidence: 99%

Exocytosis sensitivity to growth hormone-releasing hormone in subsets of GH cells in rats under different corticosterone conditions. Ultrastructural study using microwave irradiation for fixation and immunocytochemistry

Ozawa

Fang²,

Kawata³

2004

Journal of Endocrinology

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Growth hormone (GH) cells in the rat anterior pituitary have been morphologically classified into three subtypes: type I (mature) containing large secretory granules about 350 nm in diameter, type II (intermediate) containing a mixture of large and small granules, and type III (immature) containing small granules about 150 nm in diameter. However, the functional implications of morphological heterogeneity, especially the different sensitivities to growth hormone-releasing hormone (GRH) under different corticosteroid conditions have not been elucidated to date.In the present study, by application of microwave irradiation (MWI) for fixation and immunocytochemistry, new findings of the exocytotic response have been revealed among the subsets of GH cells following adrenalectomy (ADX), corticosterone treatment and/or GRH treatment.The MWI gave effective results for fixation, especially for the permeability of the fixative, and showed good results for immunoelectron microscopy using the protein-A gold method. Moreover, the use of MWI greatly shortened the fixation, processing and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling.The number of exocytotic figures was low in all subtypes of GH cells in the sham-operated control rats. GRH treatment induced a significant increase in exocytosis in each subtype of GH cells, particularly in type I (mature) and type II (intermediate) GH cells in the control rats. GRH injection to rats for 4 days after ADX also showed an increase in exocytosis, but the degree was significantly less in comparison with the GRH injection in the control group. Corticosterone replacement given to ADX rats induced a clear recovery of the exocytotic response to GRH to the control level. Serum GH content measured by radioimmunoassay correlated with these morphological results.These results suggest that the secretion of GH stimulated by GRH is closely related to corticosteroids, and that the sensitivity to GRH differs among the three subtypes of GH cells.

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Immuno-EM Localization of GFP-tagged Yolk Proteins inC. ElegansUsing Microwave Fixation

Cited by 46 publications

References 21 publications

Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

Laminin α subunits and their role inC. elegansdevelopment

Exocytosis sensitivity to growth hormone-releasing hormone in subsets of GH cells in rats under different corticosterone conditions. Ultrastructural study using microwave irradiation for fixation and immunocytochemistry

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