12 Chromosome Analysis by Fluorescence in situ Hybridization
177
Procedure
1. Select an appropriate area on the slide for hybridization and mark the Denaturation
opposite side with a diamond pen.
2. Incubate slides in an oven at 60°C prior to denaturation. This will prevent
lowering of the temperature of the denaturation solution when added to
the slides.
3. Fill denaturation solution into a Coplin jar and put it into a water bath
heated to 70°C. Check temperature of the denaturation solution with a
thermometer inside the jar. (This is a critical step!). For good results, the
temperature should be 70°C.
4. Transfer the prewarmed slides (not more than three at a time) into coplin
jar containing the denaturation solution for exactly 2 minutes.
5. Immediately transfer the slides into the Coplin jars with 70%, 90% and
100% ethanol (on ice) for 5 minutes each.
6. After air drying, the slides are ready for hybridization.
When the denaturation is not sufficient, no efficient hybridization can be
performed. Over-denatured chromosomes usually appear fuzzy when
counterstained with DAPI or may even lose their morphological features.
1. Add 10 Jll ofhybridization mixture containing the denatured probe onto Hybridization
the denatured target DNA on the slides.
2. Place an 18xl8 mm coverslip on top of the hybridization droplet. Take
care not to trap air bubbles.
3. Seal the edges of the coverslip with ruhher cement, and place the slides in
a humidified chamber. Incubate overnight at 37°C.
After hybridization, detection of the probe sequences on the slides is performed using the following protocol.
177
Procedure
1. Select an appropriate area on the slide for hybridization and mark the Denaturation
opposite side with a diamond pen.
2. Incubate slides in an oven at 60°C prior to denaturation. This will prevent
lowering of the temperature of the denaturation solution when added to
the slides.
3. Fill denaturation solution into a Coplin jar and put it into a water bath
heated to 70°C. Check temperature of the denaturation solution with a
thermometer inside the jar. (This is a critical step!). For good results, the
temperature should be 70°C.
4. Transfer the prewarmed slides (not more than three at a time) into coplin
jar containing the denaturation solution for exactly 2 minutes.
5. Immediately transfer the slides into the Coplin jars with 70%, 90% and
100% ethanol (on ice) for 5 minutes each.
6. After air drying, the slides are ready for hybridization.
When the denaturation is not sufficient, no efficient hybridization can be
performed. Over-denatured chromosomes usually appear fuzzy when
counterstained with DAPI or may even lose their morphological features.
1. Add 10 Jll ofhybridization mixture containing the denatured probe onto Hybridization
the denatured target DNA on the slides.
2. Place an 18xl8 mm coverslip on top of the hybridization droplet. Take
care not to trap air bubbles.
3. Seal the edges of the coverslip with ruhher cement, and place the slides in
a humidified chamber. Incubate overnight at 37°C.
After hybridization, detection of the probe sequences on the slides is performed using the following protocol.