1998
DOI: 10.1002/(sici)1097-0029(19980701)42:1<43::aid-jemt6>3.3.co;2-4
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Pre‐embedding immunolabeling for electron microscopy: An evaluation of permeabilization methods and markers

Abstract: For scarce antigens or antigens which are embedded in a dense macromolecular structure, on-section labeling, the first method of choice, is not always successful. Often, the antigen can be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method. Most of these methods lead to loss of ultrastructural details and, hence, labeling at electron microscope resolution does not add essential information. The scope of this paper is to compare five permeabilization methods for pre-embedding… Show more

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Cited by 16 publications
(20 citation statements)
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“…However, their use necessitates further conversion methods when subsequent electron microscopic (EM) investigations are needed to confirm their precise sites of localization (Sandell and Masland 1988;Lubke 1993;Todd 1997;Humbel et al 1998). Recently, several conversion methods have been described, including photoconversion of fluorescent tracers with DAB (Sandell and Masland 1988;Lubke 1993), the use of anti-fluorescein antibodies combined with streptavidin-gold (Shiosaka et al 1986) or immunogold (Luby-Phelps et al 1984), and the use of fluorescent tags directly (Powell et al 1997) or indirectly (Powell et al 1998) coupled with gold particles.…”
mentioning
confidence: 81%
“…However, their use necessitates further conversion methods when subsequent electron microscopic (EM) investigations are needed to confirm their precise sites of localization (Sandell and Masland 1988;Lubke 1993;Todd 1997;Humbel et al 1998). Recently, several conversion methods have been described, including photoconversion of fluorescent tracers with DAB (Sandell and Masland 1988;Lubke 1993), the use of anti-fluorescein antibodies combined with streptavidin-gold (Shiosaka et al 1986) or immunogold (Luby-Phelps et al 1984), and the use of fluorescent tags directly (Powell et al 1997) or indirectly (Powell et al 1998) coupled with gold particles.…”
mentioning
confidence: 81%
“…For electron microscopy on cells, the cells were fixed 24 h after transfection using 2% (w/v) formaldehyde and 0.25% (v/v) glutaraldehyde (EM grade; Sigma) in PBS, pH 7.4, for 30 min at room temperature. Pre-embedding labeling was performed to identify the transfected cells (19). The cells were permeabilized with 0.1% Triton X-100 and blocked with 1% bovine serum albumin fraction V, 0.045% (w/v) cold water fish gelatin (EM grade; Sigma) in PBS.…”
Section: Methodsmentioning
confidence: 99%
“…By use of this probe, the same sample can be observed at different resolutions by confocal microscopy (200-nm resolution), electron tomography (10-nm resolution), and conventional electron microscopy (1-nm resolution).The use of streptavidin-FNG before embedding greatly increases the sensitivity of antigen detection compared to classical methods using 5-10-nm gold particles on the surface of ultrathin sections (Hainfeld and Furuya 1992;Hainfeld and Powell 2000), and reveals very precisely the location and size of the structures containing antigens without altering their organization (Robinson and Vandré 1997;Baschong and Stierhof 1998;Robinson et al 2000). This detection process preserves the nuclear structure (Humbel et al 1998) and more particularly the nucleolar ultrastructure, as seen on ultrathin sections in our present and previous studies (Cheutin et al 2002).…”
Section: Figurementioning
confidence: 99%