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JULIANNE MEYNE, SUSAN M. BAILEY, and EDWIN H. GOODWIN
2. Incubate in moist chamber for the time appropriate for the probe used.
3. Remove coverslip and wash slide in 5 changes of 2xsse at 42°e. This
washing procedure works well for synthetic oligomer probes. Other
probes may require an initial high stringency wash in a formamide
solution, at the concentration used for hybridization, to reduce background fluorescence.
4. Transfer slide to a solution of PN buffer for about 5 min before counterstaining with DAPI and mounting in antifade solution.
Multiple hybridizations
Additional hybridizations can be done immediately after completing the
post-hybridization wash steps. Alternately, the slide can be mounted with
antifade and viewed under fluorescence before additional hybridizations
are done. In the latter case, the coverslip is removed and the slide is
washed in 2-3 changes of 2xsse. The slide is drained of excess sse
(but not allowed to dry) and the next hybridization mix is placed on
the slide for incubation and processing. For hybridization of singlestranded probes for both strands of a sequence, each labeled for detection
with different fluorochromes, it is often best to perform sequential hybridizations, rather than placing both probes in the same hybridization mix.
This is especially true for short repetitive sequence probes. We have frequently done 3-5 sequential hybridizations without degradation of chromosome morphology or significant increases in background.
Analysis of fluorescence
It is important to keep in mind that the target DNA is single-stranded. The
fluorescence signal may be less intense than for standard FISH using the
same probe. For most repetitive DNA sequence probes, such as alpha-satellite and classical satellite human DNA, the intensity of the fluorescence
is easily observed directly through the microscope. For other probes an
image analysis system will be advantageous.
JULIANNE MEYNE, SUSAN M. BAILEY, and EDWIN H. GOODWIN
2. Incubate in moist chamber for the time appropriate for the probe used.
3. Remove coverslip and wash slide in 5 changes of 2xsse at 42°e. This
washing procedure works well for synthetic oligomer probes. Other
probes may require an initial high stringency wash in a formamide
solution, at the concentration used for hybridization, to reduce background fluorescence.
4. Transfer slide to a solution of PN buffer for about 5 min before counterstaining with DAPI and mounting in antifade solution.
Multiple hybridizations
Additional hybridizations can be done immediately after completing the
post-hybridization wash steps. Alternately, the slide can be mounted with
antifade and viewed under fluorescence before additional hybridizations
are done. In the latter case, the coverslip is removed and the slide is
washed in 2-3 changes of 2xsse. The slide is drained of excess sse
(but not allowed to dry) and the next hybridization mix is placed on
the slide for incubation and processing. For hybridization of singlestranded probes for both strands of a sequence, each labeled for detection
with different fluorochromes, it is often best to perform sequential hybridizations, rather than placing both probes in the same hybridization mix.
This is especially true for short repetitive sequence probes. We have frequently done 3-5 sequential hybridizations without degradation of chromosome morphology or significant increases in background.
Analysis of fluorescence
It is important to keep in mind that the target DNA is single-stranded. The
fluorescence signal may be less intense than for standard FISH using the
same probe. For most repetitive DNA sequence probes, such as alpha-satellite and classical satellite human DNA, the intensity of the fluorescence
is easily observed directly through the microscope. For other probes an
image analysis system will be advantageous.