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Fluorescence and chromogenic in situ hybridization to detect genetic aberrations in formalin-fixed paraffin embedded material, including tissue microarrays

Nature Protocols volume 3, pages 220–234 (2008)Cite this article

Abstract

Screening for specific genetic aberrations by fluorescence and chromogenic in situ hybridization (fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH)) can reveal associations with tumor types or subtypes, cellular morphology and clinical behavior. FISH and CISH methodologies are based on the specific annealing (hybridization) of labeled genomic sequences (probes) to complementary nucleic acids within fixed cells to allow their detection, quantification and spatial localization. Formalin-fixed paraffin embedded (FFPE) material is the most widely available source of tumor samples. Increasingly, tissue microarrays (TMAs) consisting of multiple cores of FFPE material are being used to enable simultaneous analyses of many archival samples. Here we describe robust protocols for the FISH and CISH analyses of genetic aberrations in FFPE tissue, including TMAs. Protocols include probe preparation, hybridization and detection. Steps are described to reduce background fluorescence and strip probes for repeat FISH analyses to maximize the use of tissue resources. The basic protocol takes 2–3 d to complete.

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Figure 1
Figure 2: Demonstration of probe copy number analysis in a tissue microarray of rhabdomyosarcoma samples by chromogenic in situ hybridization (CISH) (a and c) for MYC-N (myc myelocytomatosis viral related oncogene, neuroblastoma derived) using 3,3′-diaminobenzidine (DAB) (brown) as a chromogen with hematoxylin counterstain, and by fluorescence in situ hybridization (FISH) (b and d) for FGFR1 (Fibroblast Growth Factor Receptor 1) at 8p11[green/fluorescein isothiocyanate (FITC)] with a control probe MYC-C (v-myc myelocytomatosis viral oncogene homolog) at 8q24 (red/Cy3).
Figure 3: Fluorescence in situ hybridization (FISH) detection of ERG rearrangements.

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Acknowledgements

The authors thank Sian Rizzo and Neil Goddard for critical reviewing of the manuscript.

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Authors and Affiliations

  1. Molecular Cytogenetics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG, Surrey, UK

    Brenda Summersgill & Janet Shipley

  2. Cell Transformation, Section of Molecular Carcinogenesis, The Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG, Surrey, UK

    Jeremy Clark

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Correspondence to Janet Shipley.

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Summersgill, B., Clark, J. & Shipley, J. Fluorescence and chromogenic in situ hybridization to detect genetic aberrations in formalin-fixed paraffin embedded material, including tissue microarrays. Nat Protoc 3, 220–234 (2008). https://doi.org/10.1038/nprot.2007.534

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