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

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Abstract

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.

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Authors and Affiliations

  1. Institute of Anthropology and Human Genetics, University of Munich (LMU), Munich, Germany

    Stefan Müller, Michaela Neusser & Johannes Wienberg

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  1. Stefan Müller
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  2. Michaela Neusser
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  3. Johannes Wienberg
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Müller, S., Neusser, M. & Wienberg, J. Towards unlimited colors for fluorescence in-situ hybridization (FISH) . Chromosome Res 10, 223–232 (2002). https://doi.org/10.1023/A:1015296122470

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