6 In Situ Hybridization for DNA: Fluorescent Probe
87
the hybridization mix. Place the slide with the cover slip
down on a blotter and gently press out visible bubbles.
- Hybridization conditions are inappropriate: Ensure the
temperature of the incubator is 37°C. Increase the hybridization time to 2 days. Seal the cover slip well with rubber
cement.
- Wash conditions are not correct: Ensure the temperature
and pH of washing solutions.
- If using hapten-modified probes, fluorescent staining procedures were not correct: Ensure the slides were adequately incubated with antibodies.
- Wrong counterstain used. Counterstain is too bright: Remove cover slip. Immerse slides for 5 mins in 2XSSC at
room temperature. Dehydrate the slides through a series
of 1 minute ethanol rinses (70%, 85%, 100%). Air dry
and reapply counterstain.
- Viewed hybridization using inappropriate filter sets:
Multi-bandpass filter sets provide less brightness than
the single bandpass filter sets, probe signals may appear
fainter when viewed through the multi-bandpass filter sets.
• Low signal specificity
- Too much probe in the hybridization mix: Dilute probe 10100 times.
- Inappropriate hybridization conditions: Ensure temperature of incubator is 37°C.
- Wash temperature is low: Ensure temperature of water
bath is 46°C.
- Wash solution stringency is low: Ensure the wash solutions
were made correctly.
• High slide background
- Slides were inadequately washed following hybridization:
Ensure temperature and pH of wash solutions are correct.
- If using hapten-modified probes, fluorescent staining procedures were not correct: Ensure the concentration of antibodies, often high concentration.
- Wash solutions used too long or stored improperly: Ensure
the formamide washing solution in the Coplin jars are
stored at 4°C. Discard after 1 week. Discard all other
wash solutions after 1 day. Ensure the washing solutions
are pH 7-8.
87
the hybridization mix. Place the slide with the cover slip
down on a blotter and gently press out visible bubbles.
- Hybridization conditions are inappropriate: Ensure the
temperature of the incubator is 37°C. Increase the hybridization time to 2 days. Seal the cover slip well with rubber
cement.
- Wash conditions are not correct: Ensure the temperature
and pH of washing solutions.
- If using hapten-modified probes, fluorescent staining procedures were not correct: Ensure the slides were adequately incubated with antibodies.
- Wrong counterstain used. Counterstain is too bright: Remove cover slip. Immerse slides for 5 mins in 2XSSC at
room temperature. Dehydrate the slides through a series
of 1 minute ethanol rinses (70%, 85%, 100%). Air dry
and reapply counterstain.
- Viewed hybridization using inappropriate filter sets:
Multi-bandpass filter sets provide less brightness than
the single bandpass filter sets, probe signals may appear
fainter when viewed through the multi-bandpass filter sets.
• Low signal specificity
- Too much probe in the hybridization mix: Dilute probe 10100 times.
- Inappropriate hybridization conditions: Ensure temperature of incubator is 37°C.
- Wash temperature is low: Ensure temperature of water
bath is 46°C.
- Wash solution stringency is low: Ensure the wash solutions
were made correctly.
• High slide background
- Slides were inadequately washed following hybridization:
Ensure temperature and pH of wash solutions are correct.
- If using hapten-modified probes, fluorescent staining procedures were not correct: Ensure the concentration of antibodies, often high concentration.
- Wash solutions used too long or stored improperly: Ensure
the formamide washing solution in the Coplin jars are
stored at 4°C. Discard after 1 week. Discard all other
wash solutions after 1 day. Ensure the washing solutions
are pH 7-8.