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Table 1 Troubleshooting Guidelines

From: Localization of BDNF mRNA with the Huntington's disease protein in rat brain

Problem Possible reason Solution
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.