86
MORIMASA MATS UTA AND MAYUMI MATSUTA
Dual color FISH using a combination of centromereand locus-specific probes
Nuclei
••
•
•
•
• .
.
. .. 0··
0
0 ••
0
..
•
::: .
•
••••
0
. . •
0
. .
Normal (Go/G 1)
Amplification
Amplificat!on
(Homogeneously Siaining Region) (Double Minute)
Oncogene or Tumor
• Suppressor Gene
o Centromere
Normal (G2)
Deletion
Fig. 7. Schema of deletion and amplification by dual color FISH using a combination of centromeric and locus-specific probes
2) Age slides for 24 hours at room temperature. 3) Bake
slides in the air at 6S0C for Smins.
- Target DNA are not adequately denatured: Ensure
temperature of denaturation solution (70% foramidel
2XSSC) in the Coplin jar is 70°C prior to immersing the
slide. Increase the denaturation time to 10 mins.
- Slides were not adequately processed for FISH: 1) Fix cell
specimen in methanol-acetic acid solution (3:1).2) Digest
the slides for 7.S mins in proteinase K at concentration 0.6
to 6 Ilg/m120 mM Tris / 2mM CaCh, pH 7.S at 37°C, or in
pepsin solution at concentration 0.01 to 0.S%/0.1N HCl.
Wash in 2XSSC several times, dehydrate the slides through
a series of 1 minute ethanol rinses (70%, 8S%, 100%), air
dry. Store slides at _20DC for up to 6 months.
- Probe was not adequately denatured: Ensure temperature
of the water bath used to denature the hybridization mix is
74 DC. Vortex to mix, centrifuge a few sees, and denature the
hybridization mix for S mins.
- Air bubbles were trapped under the cover slip during hybridization: Apply cover slip by first touching the surface of
MORIMASA MATS UTA AND MAYUMI MATSUTA
Dual color FISH using a combination of centromereand locus-specific probes
Nuclei
••
•
•
•
• .
.
. .. 0··
0
0 ••
0
..
•
::: .
•
••••
0
. . •
0
. .
Normal (Go/G 1)
Amplification
Amplificat!on
(Homogeneously Siaining Region) (Double Minute)
Oncogene or Tumor
• Suppressor Gene
o Centromere
Normal (G2)
Deletion
Fig. 7. Schema of deletion and amplification by dual color FISH using a combination of centromeric and locus-specific probes
2) Age slides for 24 hours at room temperature. 3) Bake
slides in the air at 6S0C for Smins.
- Target DNA are not adequately denatured: Ensure
temperature of denaturation solution (70% foramidel
2XSSC) in the Coplin jar is 70°C prior to immersing the
slide. Increase the denaturation time to 10 mins.
- Slides were not adequately processed for FISH: 1) Fix cell
specimen in methanol-acetic acid solution (3:1).2) Digest
the slides for 7.S mins in proteinase K at concentration 0.6
to 6 Ilg/m120 mM Tris / 2mM CaCh, pH 7.S at 37°C, or in
pepsin solution at concentration 0.01 to 0.S%/0.1N HCl.
Wash in 2XSSC several times, dehydrate the slides through
a series of 1 minute ethanol rinses (70%, 8S%, 100%), air
dry. Store slides at _20DC for up to 6 months.
- Probe was not adequately denatured: Ensure temperature
of the water bath used to denature the hybridization mix is
74 DC. Vortex to mix, centrifuge a few sees, and denature the
hybridization mix for S mins.
- Air bubbles were trapped under the cover slip during hybridization: Apply cover slip by first touching the surface of