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. |