6 In Situ Hybridization for DNA: Fluorescent Probe
91
5. Apply Will othybridization mix to a slide and immediately
apply 18 mm square coverslip.
6. Seal coverslip with rubber cement if hybridization time will be
greater than 2 hours.
7. Denature the probe and specimen on the slide at 70-85°C for
5 min.
8. Hybridize at 37°C for 30 mins to 2 days.
9. Wash, fluorescent stain and counterstain: Follow the subprotocols. View using a fluorescent microscope.
T rou bleshooting
The chromosome morphology observed in hybridization where
co denaturation is used may differ from a specimen that is denatured and dehydrated before the probe is applied. Often, this has
little effect on the analysis on interphase cells. Additionally,
probe targets that are over-denatured may exhibit speckling,
in which the hybridized probe appears as brightly stained spots
with smaller spots radiating from the central spots. Speckling is
more likely to occur when using higher intensity probes. Some
speckling should not interfere with chromosome enumeration.
• Cross-hybridization
- Repeat the hybridization on a new specimen using one of
the following: 1) Increase the temperature ofOAXSSC/O.3%
NP-40 by 2°C. 2) Decrease the denaturation temperature
by 2°C.
- Probe appears dim: Repeat the hybridization on a new specimen using one of the following. 1. Increase the hybridization time. 2. Increase the denaturation temperature until
the morphology becomes unacceptable. 3. Wash the slide
using OAXSSC/0.3% NP-40 at 70-73°C.
• Diffuse signal
- Repeat the hybridization on a new specimen using one of
the following. I.Decrease the denaturation temperature by
2°C. 2.Decrease the denaturation time. 3.Wash the slide
using OAXSSC/0.3% NP-40 at 73-76°C.
91
5. Apply Will othybridization mix to a slide and immediately
apply 18 mm square coverslip.
6. Seal coverslip with rubber cement if hybridization time will be
greater than 2 hours.
7. Denature the probe and specimen on the slide at 70-85°C for
5 min.
8. Hybridize at 37°C for 30 mins to 2 days.
9. Wash, fluorescent stain and counterstain: Follow the subprotocols. View using a fluorescent microscope.
T rou bleshooting
The chromosome morphology observed in hybridization where
co denaturation is used may differ from a specimen that is denatured and dehydrated before the probe is applied. Often, this has
little effect on the analysis on interphase cells. Additionally,
probe targets that are over-denatured may exhibit speckling,
in which the hybridized probe appears as brightly stained spots
with smaller spots radiating from the central spots. Speckling is
more likely to occur when using higher intensity probes. Some
speckling should not interfere with chromosome enumeration.
• Cross-hybridization
- Repeat the hybridization on a new specimen using one of
the following: 1) Increase the temperature ofOAXSSC/O.3%
NP-40 by 2°C. 2) Decrease the denaturation temperature
by 2°C.
- Probe appears dim: Repeat the hybridization on a new specimen using one of the following. 1. Increase the hybridization time. 2. Increase the denaturation temperature until
the morphology becomes unacceptable. 3. Wash the slide
using OAXSSC/0.3% NP-40 at 70-73°C.
• Diffuse signal
- Repeat the hybridization on a new specimen using one of
the following. I.Decrease the denaturation temperature by
2°C. 2.Decrease the denaturation time. 3.Wash the slide
using OAXSSC/0.3% NP-40 at 73-76°C.