Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization.
A new method was developed to prepare and isolate interphase nuclei from murine preimplantation embryos for analysis by fluorescence in-situ hybridization. Embryos were washed with phosphate-buffered saline and disaggregated in a small drop of bidistilled water containing HCl and Tween 20. During the disaggregation procedure embryos were watched continuously with an inverted microscope. Embryonic nuclei were digested with pepsin to make them accessible for hybridization to the probes and to remove remnants of cytoplasm. Nuclear and probe DNA were denatured simultaneously and hybridization was done overnight, followed by immunocytochemical detection of the probes. Using this method we were able to perform successful in-situ hybridization on all preimplantation stages of the mouse embryo (pronuclei, 2-cell, 4-cell, morula and blastocyst). Probes for the X and Y chromosomes were applied for sex determination. From the results described in this paper we conclude that the preparation and isolation of interphase nuclei from murine embryos with acid and Tween 20 offers high reproducibility, good morphology of the cells and a high hybridization efficiency.
Coonen
E
E
Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands.
Dumoulin
J C
JC
Ramaekers
F C
FC
Hopman
A H
AH
D000818:Animals; D001755:Blastocyst / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000737:chemistry* / Q000648:ultrastructure; D004247:DNA / Q000032:analysis; D015342:DNA Probes; D005260:Female; D006851:Hydrochloric Acid; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009028:Morula / Q000648:ultrastructure; D011136:Polysorbates; D014960:X Chromosome; D014998:Y Chromosome
D015342:DNA Probes; D011136:Polysorbates; D004247:DNA; D006851:Hydrochloric Acid