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Methods Cell Biol. 2018;144:287-305. doi: 10.1016/bs.mcb.2018.03.015.

Analysis of chromosomes from mouse oocytes and mammalian cultured cells by light microscopy.

Author information

1
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.
2
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria.
3
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria. Electronic address: jan-michael.peters@imp.ac.at.

Abstract

As carriers of the genetic material, chromosomes are of prime interest in the life sciences. Although all aspects of chromosome biology should ideally be studied in living cells, the isolation of chromosomes can greatly facilitate their analysis. This can be achieved by lysing mitotic or meiotic cells under conditions where their content, including their chromosomes, is spread out on the surface of microscopy glass slides. Here we describe three such chromosome spreading techniques, which have been instrumental in analyzing chromosomes from either mouse oocytes or mammalian cultured cells in mitosis. For both chromosomes from oocytes and mitotic cells, we describe immunofluorescence protocols that enable the visualization of proteins with specific antibodies. For mitotic chromosomes, we also provide a classic protocol for Giemsa staining. This protocol cannot be used to localize proteins but is useful to determine structural features of chromosomes, such as sister chromatid cohesion and chromosome condensation. The question of how chromosome nondisjunction during the meiotic division causes aneuploidy is of great interest in oocyte chromosome research. Because we have found that ploidy in mouse oocytes can be determined more reliably in fixed cells than in spread chromosomes, we also describe a protocol for the in situ fixation and immunofluorescence analysis of chromosomes in mouse oocytes.

KEYWORDS:

Chromosome spreading; Chromosomes; Immunofluorescence; Mammalian cultured cells; Oocytes

PMID:
29804673
DOI:
10.1016/bs.mcb.2018.03.015
[Indexed for MEDLINE]

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