29 Spectral Imaging Analysis of Metaphase and Interphase Cells
377
7. Add 7 ~l of hybridization master mix after the pellet is totally dissolved. This gives a total hybridization mixture of 10 ~l.
8. Store the hybridization mixture at -20°C until use.
Caution: Often one fluorochrome shows a stronger signal compared to
another fluorochrome when bound to the same DNA. For example, the
intensity of probes detected with CyS.5 is usually much stronger than
the intensity of Cy3 labeled probes. This effect is probably due to a combination of different quantum efficiencies, probe labeling efficiencies, energy transfer and photon detection sensitivity. To avoid masking the Cy3
fluorescence with CyS or CyS.5 emission, Cy3 probes are used at a higher
concentration. Therefore, the composition of probes is very important for
the interphase cell analysis by FISH when using the Spectral Imaging system.
Subprotocol 3
Hybridization and detection of bound probes
Procedure
These procedures follow the protocol provided in the SKY kit.
1. The hybridization mixture (vial-l of SKY kit or locus-specific probes)
is denatured at 76°C for 7 min, then allowed to pre-anneal at 37°C for
60 min.
2. Slides are denatured in denaturing solution at 76°C for 2-5 min. The
denaturing time for slides depends on the different types of slides.
3. Then, slides are dehydrated in 70%,80%, and 100% ethanol for 2 min
each step, and allowed to air dry.
4. The hybridization mixture is applied to the slides under a glass coverslip, which is sealed with rubber cement. Finally, the hybridization proceeds at 37°C for 40-48 h in a moisture chamber.
5. Following hybridization, wash slides at 43°C three times in 50% FAI
2x SSC for 10 min each, followed by two washes in 2x SSC for 10
min each and one wash in 0.4 x SSC for 5 min.
377
7. Add 7 ~l of hybridization master mix after the pellet is totally dissolved. This gives a total hybridization mixture of 10 ~l.
8. Store the hybridization mixture at -20°C until use.
Caution: Often one fluorochrome shows a stronger signal compared to
another fluorochrome when bound to the same DNA. For example, the
intensity of probes detected with CyS.5 is usually much stronger than
the intensity of Cy3 labeled probes. This effect is probably due to a combination of different quantum efficiencies, probe labeling efficiencies, energy transfer and photon detection sensitivity. To avoid masking the Cy3
fluorescence with CyS or CyS.5 emission, Cy3 probes are used at a higher
concentration. Therefore, the composition of probes is very important for
the interphase cell analysis by FISH when using the Spectral Imaging system.
Subprotocol 3
Hybridization and detection of bound probes
Procedure
These procedures follow the protocol provided in the SKY kit.
1. The hybridization mixture (vial-l of SKY kit or locus-specific probes)
is denatured at 76°C for 7 min, then allowed to pre-anneal at 37°C for
60 min.
2. Slides are denatured in denaturing solution at 76°C for 2-5 min. The
denaturing time for slides depends on the different types of slides.
3. Then, slides are dehydrated in 70%,80%, and 100% ethanol for 2 min
each step, and allowed to air dry.
4. The hybridization mixture is applied to the slides under a glass coverslip, which is sealed with rubber cement. Finally, the hybridization proceeds at 37°C for 40-48 h in a moisture chamber.
5. Following hybridization, wash slides at 43°C three times in 50% FAI
2x SSC for 10 min each, followed by two washes in 2x SSC for 10
min each and one wash in 0.4 x SSC for 5 min.