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FISH 'n' Tips: Sample Preparation

Section 2 Preperation

View our collection of FISH 'n' Tips for the sample preparation stage of the fluorescence in situ hybridization (FISH) protocol. Tips include how to optimize tissue enzyme digestion results, why you should avoid baking or aging of slides, and advice on improving cell morphology. 

Steve Chatters, Acting Head, Regulatory and Medical Afairs

Steve Chatters

When using the enzyme solution to digest your FFPE slide, liberally apply the enzyme ensuring the section is covered, as evaporation may cause the enzyme to recede from the edges and cause inconsistent digestion.

Jennifer Golding, Global Product Manager - FISH

Jen Golding white background

If your cells appear intact with a three-dimensional look to them, and there is visible cytoplasm using a phase contrast microscope, you could try re-fixing your cells in 1:1 methanol:acetic acid.

Daniela Johnanes, FISH Field Application Support, Europe

Daniela v2

Try pre-fixing, your bone marrow or peripheral blood samples by slowly adding ice-cold fix with agitation, immediately after the hypotonic treatment, to improve your cell preparations.

Ashley Hart, FISH Field Application Scientist, US & Canada

Ashley v2

Consider using a mild hypotonic treatment before fixing CD138+ selected cells, which may improve cell morphology.

Faidra Partheniou, Medical Affairs Manager

Faidra

Positively charged slides should only be used for FFPE sections. If these slides are used for cell suspensions, they may produce high green background.

Gothami Fonseka, FISH Field Application Support, Europe

Gothami v2

After pre-treatment, consider using DAPI to assess for over/under digestion of FFPE sections; this can be washed off before applying the FISH probe.

Faidra Partheniou, Medical Affairs Manager

Faidra

When using archived cell pellets, consider re-fixing using fresh fixative to ensure they provide optimal results.

Jennifer Golding, Global Product Manager - FISH

Jen Golding white background

Consider using Pepsin to digest excess cytoplasm and cellular debris in Cytospin samples to improve hybridization quality.

Ashley Hart, FISH Field Application Scientist, US & Canada

Ashley v2

Try cleaning your slides in 70% ethanol before use, to get rid of any dust or debris. A high degree of debris may lead to a high background.

Daniela Johnanes, FISH Field Application Support, Europe

Daniela v2

Try to avoid baking or aging of slides (if not needed for another application e.g. G-Banding) as it may reduce signal fluorescence.

Steve Chatters, Acting Head, Regulatory and Medical Afairs

Steve Chatters

Perform tissue enzyme digestion at 37°C on a hotplate for best results.

Gothami Fonseka, FISH Field Application Support, Europe

Gothami v2

When performing enzyme digestion, why not try using Parafilm® to cover the area. This may aid spreading of the reagents and reduces enzyme evaporation.