Background staining during in situ hybridization (ISH) may be excessive for a number of reasons. The problem has to be identified before it can be corrected.
Reasons for High Background Stain
Solution
The blocking step was skipped.
Make sure that the blocking step is done.
The probe is too concentrated.
Use the optimum probe concentration.
The slides were allowed to dry out during incubations.
Do not let the slides dry out.
One or more wash steps were not done adequately.
Perform adequate washing.
The detergent wash buffer was not used after label incubation.
Use the detergent wash buffer after label incubation.
An incubation was too long.
Do not prolong the incubation step.
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