Demonstration of chromosome replication by BrdU antibody technique and electron microscopy

Abstract

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.

Silver Staining of Selected Mitoses by Physical Development

“…It can be used also in combination with C-banding [8]. Vogel et al [9] reported the use of silver enhancement enabling easy detection of immunogold particles. Fontana and Goldini [10] used the silver staining method of Howell and Black [3] to demonstrate banding pattern on prophase and promethaphase male termite chromosomes induced by actinomycin D. The earlier silver staining methods applied silver impregnation and a separate solution of a developing agent.…”

Fiber-Like Organization as a Basic Principle for Euchromatin Higher-Order Structure

“…The ability to discern label distribution with bright-field optical microscopy greatly facilitated preselection of nuclei with labeled euchromatin for sectioning and tomography. This protocol has several advantages over previously published ones (Vogel et al, 1990;Jaunin and Fakan, 2002;Philimonenko et al, 2004;Koberna et al, 2005;Deng et al, 2016). Glutaraldehyde fixation ensures optimal preservation of chromatin near-native structure.…”

Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites andin situ hybridization

“…Latos-Bielenska et al (1987) found the two staining techniques comparable but the immunohistochemical technique stained a few GTG bands and constitutive chromatin less intensely. Latos-Bielenska and Vogel (1990Vogel ( , 1992 observed that heterochromatin was mostly synthesized in early S-phase, with most of the synthesis before the initiation of G-banding. Furthermore, they found that the immunohistochemical method was as sensitive as the [3H]dThd incorporation, and more sensitive than the traditional Giemsa differential staining of chromosome bands.…”