Molecular markers
Fluorescent in situ hybridization (FISH)
FISH protocol




Topic Completed: 1 April 2012

Revised: 15 February 2019

Copyright: 2003-2018, PathologyOutlines.com, Inc.

PubMed Search: FISH protocol[TIAB]

Page views in 2018: 224
Page views in 2019 to date: 62
Table of Contents
Definition / general
Cite this page: Shackelford R. FISH protocol. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/molecularpathfishprotocol.html. Accessed April 18th, 2019.
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)
Back to top