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

Nat Protoc. 2008;3(2):220-34. doi: 10.1038/nprot.2007.534.


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.

MeSH terms

  • Chromogenic Compounds
  • Chromosome Aberrations*
  • Formaldehyde
  • In Situ Hybridization / methods*
  • In Situ Hybridization, Fluorescence / methods
  • Paraffin Embedding
  • Proto-Oncogene Proteins c-myc / analysis
  • Rhabdomyosarcoma / genetics
  • Serine Endopeptidases / analysis
  • Tissue Array Analysis / methods
  • Tissue Fixation
  • Trans-Activators / analysis
  • Transcriptional Regulator ERG


  • Chromogenic Compounds
  • ERG protein, human
  • Proto-Oncogene Proteins c-myc
  • Trans-Activators
  • Transcriptional Regulator ERG
  • Formaldehyde
  • Serine Endopeptidases
  • TMPRSS2 protein, human