Tips include how to avoid creating a patchy hybridization, why it's a good idea to clean your slides in 70% ethanol before use, and why long pipette tips can help prevent cross contamination.
Watch our video on how to prevent patchy hybridization during the probe application stage of the FISH protocol.
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.
Laurence Cambridge
FISH Field Application Specialist, Europe
To avoid patchy hybridizations, make sure to press out visible bubbles when applying the coverslip on the probe mix.
Ashley Hart
FISH Field Application Scientist, North America
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.
Jenny Morse
FISH Field Application Scientist, North America
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.
Graham Halford
FISH Field Application Specialist, Europe
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.
Ashley Hart
FISH Field Application Scientist, North America
If your chromosomes appear distorted this may be because the coverslip was not sealed. In order to improve chromosome morphology, try sealing the coverslips prior to hybridization.
Graham Halford
FISH Field Application Specialist, Europe
Filtered pipette tips can reduce intermittent background issues from debris being expelled onto FISH slides.
Laurence Cambridge
FISH Field Application Specialist, Europe
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.
Jenny Morse
FISH Field Application Scientist, North America
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