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FISH 'n' Tips: Probe Application

Section 3 Probe application

View our collection of FISH 'n' Tips for the probe application stage of the fluorescence in situ hybridization (FISH) protocol. Tips include how to avoid creating a patchy hybridization, why its a good idea to clean your slides in 70% ethanol before use, and why long pipette tips can help prevent cross contamination.

Gothami Fonseka, FISH Field Application Support, Europe

Gothami v2

Try not to push down too hard on the coverslip when applying the FISH probe, as it can squeeze out of the sides and create a patchy hybridization.

Ashley Hart, FISH Field Application Scientist, US & Canada

Ashley v2

To avoid patchy hybridizations, make sure to press out visible bubbles when applying the coverslip on the probe mix.

Daniela Johnanes, FISH Field Application Support, Europe

Daniela 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.

Steve Chatters, Acting Head, Regulatory and Medical Afairs

Steve Chatters

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.

Faidra Partheniou, Medical Affairs Manager

Faidra

Ensure pipette tips are long enough so that when the tip reaches the bottom of the vial, the pipette itself doesn’t come into contact. Pipettes entering a probe vial may carry over probe reagent from one vial to the next resulting in cross contamination.

Jennifer Golding, Global Product Manager - FISH

Jen Golding white background

If your chromosomes appear distorted this may be because the coverslip was not sealed. By sealing the coverslips prior to hybridization, this improves the overall chromosome morphology.

Gothami Fonseka, FISH Field Application Support, Europe

Gothami v2

Filtered pipette tips can reduce intermittent background issues from debris being expelled onto FISH slides.

Daniela Johnanes, FISH Field Application Support, Europe

Daniela v2

When making FISH slides from fixed cell suspensions, use a template to ensure suspensions are consistently spotted in the same place. Use the template for probe application also, which ensures the FISH probe is applied in the correct place.