|No or weak hybridization signal||Probe degradation||
Check probe on DNA gel.|
If necessary, synthesize new probe.
|Probe concentration too low||
Use more probe.|
Use longer hybridization time.
Optimize perfusion and post-fixation conditions.|
Ensure that solutions, containers and instruments are RNase-free.
|Inadequate immunofluorescent detection of DIG-labeled probes||
Titrate anti-hapten antibody.|
Replace with new antibody.
Replace with more sensitive detection system (e.g. polyclonal anti-hapten).
Use brighter fluorescent dye conjugated antibodies (e.g. Dylight).
|FISH signal detected on section surface only||Poor permeabilization||
Use longer incubation time or optimize detergent concentration.|
Use thinner sections.
Use shorter probe or oligonucleotide probe.
|Non-specific and/or high background staining||Probe not well purified||Optimize purification steps.|
|Hybridization conditions not optimal||
Reduce time of hybridization.|
Reduce probe amount.
Increase time and volume of post-hybridization washes.
Increase concentration of serum in blocking steps.
Use 4°C for incubation with secondary antibodies.
Use gentle shaking for hybridization, washing steps.
Avoid sections drying out.
|Hybridization of probes to unwanted mRNAs||
Use coding sequence only instead of entire plasmid or shorter sequence for probe generation.|
Use sequence-specific probes.
Use oligonucleotide probes.
|Immunofluorescent detection of DIG-labeled probes not optimal||
Adjust concentration of anti-DIG and/or anti-chicken antibodies.|
Spin antibody before use.
Use another detection system.