Haematology FISH protocol

1

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.

2

Pre-denaturation

• 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.

3

Denaturation

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

4

Hybridisation

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

5

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.

6

Analyse

• 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.