Multiplex-fluorescence in situ hybridization for chromosome karyotyping

Nat Protoc. 2006;1(3):1172-84. doi: 10.1038/nprot.2006.160.


Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes--the 22 autosomes and X and Y sex chromosomes--with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by multichannel image-analysis methods. Advantages of M-FISH include rapid analysis of metaphase spreads, even in complex cases with multiple chromosomal rearrangements, and identification of marker chromosomes. The M-FISH technology has been extended to other species, such as the mouse. Furthermore, in addition to painting probes, the method has been used with a variety of region-specific probes. M-FISH has even recently been used for 3D studies to analyze the distribution of human chromosomes in intact and preserved interphase nuclei. Hence, M-FISH has evolved into an essential tool for both clinical diagnostics and basic research. In this protocol, we describe how to use M-FISH to karyotype chromosomes, a procedure that takes approximately 14 d if new M-FISH probes have to be generated and 3 d if the M-FISH probes are ready to use.

MeSH terms

  • Animals
  • Chromosomes / genetics*
  • Fluorescent Dyes / analysis
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • Karyotyping / methods
  • Mice


  • Fluorescent Dyes