First slide
Haematology FISH protocol Image

Sample and slide preparation

  • Spot the cell sample onto a glass microscope slide. Allow to dry.
  • Immerse the slide in 2x Saline Sodium Citrate (SSC) for 2 minutes at room temperature (RT) without agitation.
  • Dehydrate in an ethanol series (70%, 85% and 100%), each for 2 minutes at RT.
  • Allow to dry.
Sample and slide preparation Image


  • Remove the probe from the freezer and allow it to warm to RT. Briefly centrifuge tubes before use.
  • Ensure that the probe solution is sufficiently mixed with a pipette or a vortex mixer.
  • Remove 10μl of probe per test, and transfer it to a microcentrifuge tube. Quickly return the remaining probe to -20ºC.
  • Place the probe and the sample slide to prewarm on a 37°C (+/- 1°C) hotplate for 5 minutes.
  • Spot 10μl of probe mixture onto the cell sample and carefully apply a 24x24mm coverslip. Seal with rubber solution glue and allow the glue to dry completely.
Pre-denaturation Image


  • Denature the sample and probe simultaneously by heating the slide on a hotplate at 75°C (+/- 1°C) for 2 minutes.
Denaturation Image


  • Place the slide in a humid, lightproof container at 37°C (+/- 1°C) overnight.
Hybridisation Image

Post-hybridisation washes

  • Remove the DAPI from the freezer and allow it to warm to RT.
  • Remove the coverslip and all traces of glue carefully.
  • Immerse the slide in 0.4x Saline Sodium Citrate (SSC) (pH 7.0) at 72°C (+/- 1°C) for 2 minutes without agitation.
  • Drain the slide and immerse it in 2xSSC + 0.05% Tween-20 at RT (pH 7.0) for 30 seconds without agitation.
  • Drain the slide and apply 10μl of DAPI antifade onto each sample.
  • Cover with a 24x24mm coverslip, remove any bubbles.
  • Edge the slide with clear nail varnish to seal.
  • Allow the colour to develop in the dark for 10 minutes.
Post-hybridisation washes Image


  • View with a fluorescence microscope.
  • For optimal visualisation of the probes, a 100-Watt mercury lamp (or equivalent) is recommended with plan apochromat objectives 63x or 100x.
  • Filters designed specifically for detection of DAPI, FITC, Texas Red®, and Aqua or DEAC fluorophores individually or in combination (e.g. dual or triple filters) are optimal for best results.
Analyse Image
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