Towards unlimited colors for fluorescence in-situ hybridization (FISH)

Chromosome Res. 2002;10(3):223-32. doi: 10.1023/a:1015296122470.


We describe a FISH protocol that allows rehybridization of complex DNA probes up to four times to the same specimen. This strategy, which we termed ReFISH, opens a wide range of new applications to conventional band pass filter epifluorescence microscopy. These include M-FISH karyotyping and cross-species color banding that emulate multiplex probe sets labeled with up to 12 fluorochromes in sequential hybridizations to the same specimen. We designed a human 24-color karyotyping probe set in combination with a 29-color cross-species color banding probe set using gibbon painting probes. Applying the ReFISH principle, 53 painting probes on individual metaphases were discriminated. This allowed simultaneous screening for inter- and intrachromosomal rearrangements on normal human diploid cells, a HeLa derived cell line, and highly rearranged gibbon chromosomes. Furthermore, the present ReFISH experiments successfully combine 24-color FISH with laser scanning confocal microscopy to study the 3D organization of all 46 human chromosome territories in individual interphase cell nuclei.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Cell Line
  • Chromosome Banding
  • Chromosome Painting / methods*
  • Color
  • HeLa Cells
  • Humans
  • Hylobates / genetics
  • Karyotyping
  • Lymphocytes / ultrastructure
  • Microscopy, Confocal
  • Species Specificity