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THE FISH assay resulted in high slide background

Was slide cleaned properly prior to sample preparation? 

-Immerse slide in ethanol, dry using lint-free paper 

Were the Specimen slides used too soon prior to denaturation? 

-Ensure slides are aged at least 24 hours at ambient temperature 

Could there have been too much cellular debris on the sample? 

-Wash cell pellet with fresh fixative three times prior to dropping slides 

Were the metaphase spreads aged by baking, or do they contain cytoplasm? 

-Increase time the slide is immersed in the denaturation solution to 10 minutes 

Was the slide inadequately washed following hybridization? 

-Ensure wash solutions were made according to procedures outlined by the package insert 

-Make sure the wash solution is at the proper PH and temperature, remove cover slip and repeat the wash procedure 

-Increase immerse time to 4 minutes in the 73°C 0.4XSSC/0.3% NP-40 wash 

Were the wash solutions used too long or improperly stored? 

-Ensure wash solutions containing formamide are properly stored at 4°C 

-Discard these solutions after 7 days or after frequent use; discard all other solutions after 1 day 

-Ensure the pH of formamide solutions are pH 7.0-8.0 

Was the hybridization viewed using long bandpass filters? 

- Reduce background light by switching to filters with smaller bandwidths or to multi-bandpass filters


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