Electron microscopy of differentially BrdU-substituted sister chromatids treated with sodium phosphate

Burkholder, Gary D.; Wang, H. C.

doi:10.1007/bf00292219

Cited by 12 publications

(3 citation statements)

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“…Taking the average of six cells each, we found that the increase of electron density in the chromosomal DNA area was approximately 30% in cells incubated for 12 h, and 45% in cells incubated for 24 h in HDC TEM images. Previous attempts at visualization of BrdU‐DNA in thick samples were unsuccessful (Burkholder & Wang, 1978). It is likely that the reasons why BrdU‐containing DNA could be recognized through its electron density alone in this study are the absence of staining (except for BrdU itself) and the higher accelerating voltage of 300 kV.…”

Section: Resultsmentioning

confidence: 99%

Visualization of BrdU‐labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

Nitta¹,

Nagayama

Danev

et al. 2009

Journal of Microscopy

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SummaryWe have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.

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Section: Resultsmentioning

confidence: 99%

Visualization of BrdU‐labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

Nitta¹,

Nagayama

Danev

et al. 2009

Journal of Microscopy

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“…BrdU substitution of chromosomal segments or chromatids can be seen directly on transmission EM (Burkholder and Wang 1978), though the contrast usually is poor. Detection of BrdU incorporation by silver staining (Ag NOR, Goodpasture and Bloom 1975) is not specific enough (Vogel et al 1978), but can be used for EM (Goyanes and Schvartzman 1983).…”

Section: Introductionmentioning

confidence: 98%

Demonstration of chromosome replication by BrdU antibody technique and electron microscopy

1990

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We describe the use of different reporter groups in visualizing replication patterns on metaphase chromosomes after BrdU incorporation by the BrdU antibody technique for the electron microscope. There is an inverse correlation between the density of the label and the size of the reporter particles. This observation alludes to stereo problems interfering with the access of the labeled antibodies into the chromatin. The use of silver enhancement enables easy detection of 1-nm and 5-nm gold particles, which make replication patterns visible in the electron microscope as does the diaminobenzidine/H2O2 reaction. Possible consequences for the demonstration of replication patterns and for nonradioactive DNA in situ hybridization are discussed.

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“…However, the few attempts that have been made so far (Binder and Kim. 1975: Burkholder andWang, 1978;Takayama et al, 1981: Utsumi. 1982) have failed to provide new insights, mainly because sister chromatids containing different amounts of BrdU could not be differentiated without disrupting the structure of the chromosomes themselves.…”

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confidence: 99%

Electron microscopy of sister chromatid exchanges

Goyanes

Schvartzman

1983

Cytogenet Genome Res

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BrdU-substituted chromosomes of Don cells were differentially stained with ammoniacal silver carbonate and analyzed by electron microscopy. The results obtained led us to the following conclusions: (1) the differential impregnation of BrdU-substituted chromatids with silver is mainly due to a preferential extraction of non-histone proteins caused by UV-irradiation and incubation in SSC at 55 °C prior to silver staining; (2) the core-like structures detected after silver staining may well represent an artifact due to the effects of severe hypotonic pre-treatment on chromatin dispersion; (3) in each sister-chromatid exchange, the border of exchange clearly defines the course of DNA fibers within the chromatid. This border of exchange tends to run perpendicular to the chromosome axis but may vary slightly depending on the degree of chromosome condensation.

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Electron microscopy of differentially BrdU-substituted sister chromatids treated with sodium phosphate

Cited by 12 publications

References 11 publications

Visualization of BrdU‐labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

Visualization of BrdU‐labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

Demonstration of chromosome replication by BrdU antibody technique and electron microscopy

Electron microscopy of sister chromatid exchanges

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