Molecular markers
Fluorescent in situ hybridization (FISH)
FISH protocol

Topic Completed: 1 April 2012

Minor changes: 15 February 2019

Copyright: 2003-2018,, Inc.

PubMed Search: FISH protocol[TIAB]

Rodney E. Shackelford, D.O., Ph.D.
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Table of Contents
Definition / general
Cite this page: Shackelford R. FISH protocol. website. Accessed May 27th, 2020.
Definition / general
  • Goal is to identify targets with high specificity, although research projects seeking related sequences intentionally use lower specificity
  • FISH employs three sequential steps:
    1. Sample fixation
    2. Probe denaturation and hybridization
    3. Several washing steps
  • Fixation:
    • Requires that sample is fixed to microscope slide so it does not wash off but loose enough to allow probe target hybridization
  • Probe denaturation and hybridization:
    • Usually marked excess of probe to target sequence is used to insure complete target binding
    • Stringency of binding is manipulated using salt and detergent concentrations, temperature and hybridization time
    • Hybridization volumes are relatively low (10 - 20 ul), to minimize volume of expensive probes
  • Hybridization temperatures are typically 37 - 60 °C in buffers containing 50% formamide and SSC buffer
  • Tissue samples are usually pretreated with proteases, appropriate nucleases and other reagents to remove sample constituents that might interfere with FISH (examples - RNase pretreatment to remove RNA from hybridization to DNA, proteolysis of DNases or RNases to avoid probe degradation, DNase pretreatment to remove DNA from reactions hybridizing probe to sample RNA)
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