FISH on 3D preserved bovine and murine preimplantation embryos.
Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of whole chromosomes or subchromosomal regions, such as chromosome arms, bands, centromeres, or single gene loci. FISH is routinely performed on chromosome spreads, as well as on three-dimensionally preserved cells or tissues (3D FISH). We have developed 3D FISH protocol for mammalian preimplantation embryos to investigate the nuclear organization of chromosome territories and subchromosomal regions during the first developmental stages. In contrast to cells, embryos have much more depth and their nuclei are therefore less accessible to probes used to visualize specific genomic regions by FISH. The present protocol was developed to establish a balance between sufficient embryo permeabilization and maximum preservation of nuclear morphology.
Koehler
Daniela
D
Institute of Human Genetics, Biozentrum (LMU), University of Münich, Planegg-Martinsried, Germany. Dani.Koehler@lrz.uni-muenchen.de1
Zakhartchenko
Valeri
V
Ketterl
Nina
N
Wolf
Eckhard
E
Cremer
Thomas
T
Brero
Alessandro
A
D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism; D002417:Cattle; D021621:Imaging, Three-Dimensional; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008856:Microscopy, Fluorescence; D009691:Nucleic Acid Denaturation; D016610:Tissue Embedding; D016707:Tissue Fixation; D014021:Tissue Preservation / Q000379:methods*