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FISH protocol: Probe application

Can I reduce or dilute the probe volume prior to application?

Cytocell probes (aside from Sub-telomeric and enumeration probes) are supplied ready to use with probe and hybridisation solution pre-mixed. This mix has been developed and optimised for the intended purpose using 10μl of probe and a 24mmx24mm coverslip. For this reason, we do not recommend dilution or reduction in volume. Both can produce weaker, more variable probe signals.

Can you use an alternative to a glass coverslip?

Recommendations:

- Use a glass coverslip. Avoid using alternatives such as parafilm, clingfilm etc. These will not create a sufficient seal to allow for probe spreading.

- Ensure coverslips are sealed to prevent evaporation which can lead to weak, diffuse green signals.

- Seal correctly with rubber glue solution e.g. Weldite/Fixogum

How can I reduce the risk of probe contamination when using different probes during my preparation?

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.