Table 1 Troubleshooting Guidelines

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.

mRNA degradation

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.