FFPE FISH protocol

Slide preparation

For FISH, 4μm - 6μm thick FFPE tissue sections should be used. Slides should be treated with an adhesive before mounting tissue section. Throughout the entire procedure, unless otherwise indicated, it is important that the tissue section does not dehydrate. For optimal results, use the Tissue Pretreatment Kit (LPS 100).

Heat pretreatment

Heat 50ml Tissue Pretreatment Solution (Reagent 1) in a porcelain wash jar or coplin jar immersed in a waterbath until it is either boiling or 98 - 100°C.
Boil slides for 30 minutes (Note: different incubation times may be required depending on tissue fixation. A 30-minute incubation is a recommended starting point).
Wash in PBS or dH₂O at room temperature (RT) for 2 x 3 minutes.

Enzyme digestion

Cover tissue with 100-200μl of Enzyme Reagent (Reagent 2) for 10 minutes at RT. (Note: depending on tissue fixative used, different incubation times may be required. Excessive digestion will cause loss of nuclei and chromosome structure.)
Wash in PBS or dH₂O at RT for 3 x 2 minutes.
Dehydrate slides in a series of 70%, 85%, 95% and 100% ethanol for 2 minutes each at room temperature, air dry, and proceed to denaturation and hybridisation.

Pre-denaturation

Remove the probe from the freezer and allow it to warm to room temperature (RT).
Ensure that the probe solution is uniformly mixed with a pipette.
Remove 10μl - 15μl (depending on the size of the tissue) of probe per test, and transfer it to a microcentrifuge tube. Quickly return the remaining probe to -20°
Place the probe and the sample slide to prewarm on a 37°C (+/- 1°C) hotplate for 5 minutes.
Spot 10μl - 15μl of probe mixture onto the sample and carefully apply a coverslip. Seal with rubber solution glue and allow the glue to dry completely.

Denaturation

Denature the sample and probe simultaneously by heating the slide on a hotplate at 75°C (+/- 1°C) for 5 minutes.

Hybridisation

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

Post-hybridisation washes

Remove the coverslip and all traces of glue carefully.
Immerse the slide in 0.4xSSC (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 - 15μl of DAPI antifade onto each sample.
Cover with a coverslip, remove any bubbles and allow the colour to develop in the dark for 10 minutes.

Analyse

View with a fluorescence microscope.

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FFPE tissue preparation and FISH protocol