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BURT G. FEUERSTEIN et aI.
3. Wash slides by immersing for 10 min successively in 4x SSC, 4x SSC/
0.1% Triton X-toO, and again in 4x sse. Immerse for 5 min each in two
changes of PN and air dry.
Mounting 1. Apply - 25 III of DAPI mounting solution per slide and drop a coverslip
that covers the whole slide, taking care to expel any air. Coverslips
should be stored in methanol and dried with a kimwipe or cleaned
immediately before use.
2. The completed slides can be cleaned but the coverslip should not be
disturbed. To clean slides, fold a paper towel in thirds. Place slides in
the center of the towel, fold the edges over the slide, and blot excess
DAPI by pressing on the covered slides. Constant pressure kept on one
end of the coverslip will prevent movement. Then, using a kimwipe to
tightly hold one end of the coverslip on the slide, fog the slide by
breathing on it and wipe clean with another kimwipe. Repeat with
the reverse side of the slide, and store them. Slides are now ready
for image acquisition.
Results
Microscopy and imaging
Visual inspection It is important to directly visualize CGH hybridizations using an appropriately equipped fluorescence microscope. Collecting images from poor
quality hybridizations are, at best, a waste of time. At worst, they may
generate erroneous results. The experimenter should choose four to six
metaphases per experiment with relatively equivalent condensation, excellent spreading (minimal bending, touching, or overlapping chromosomes), negligible fluorescent background, smooth fluorescence distributed along the chromosome, and good DAPI chromosome banding.
Good DAPI banding is critical for chromosome identification. If the
chromosomes are overdenatured or overdigested, hybridization may be
excellent, but the chromosomes may be unrecognizable. Therefore, the
experiment should balance between good hybridization and easy identification of chromosomes.
Excellent blocking of repeat sequences in chromosomes by CoT-1 DNA
is crucial (I 1). The goal of a CGH experiment is to measure a hybridization
that reflects the copy number of unique sequences in the test and reference
DNAs. If repeat sequences on the metaphase chromosomes are not well
BURT G. FEUERSTEIN et aI.
3. Wash slides by immersing for 10 min successively in 4x SSC, 4x SSC/
0.1% Triton X-toO, and again in 4x sse. Immerse for 5 min each in two
changes of PN and air dry.
Mounting 1. Apply - 25 III of DAPI mounting solution per slide and drop a coverslip
that covers the whole slide, taking care to expel any air. Coverslips
should be stored in methanol and dried with a kimwipe or cleaned
immediately before use.
2. The completed slides can be cleaned but the coverslip should not be
disturbed. To clean slides, fold a paper towel in thirds. Place slides in
the center of the towel, fold the edges over the slide, and blot excess
DAPI by pressing on the covered slides. Constant pressure kept on one
end of the coverslip will prevent movement. Then, using a kimwipe to
tightly hold one end of the coverslip on the slide, fog the slide by
breathing on it and wipe clean with another kimwipe. Repeat with
the reverse side of the slide, and store them. Slides are now ready
for image acquisition.
Results
Microscopy and imaging
Visual inspection It is important to directly visualize CGH hybridizations using an appropriately equipped fluorescence microscope. Collecting images from poor
quality hybridizations are, at best, a waste of time. At worst, they may
generate erroneous results. The experimenter should choose four to six
metaphases per experiment with relatively equivalent condensation, excellent spreading (minimal bending, touching, or overlapping chromosomes), negligible fluorescent background, smooth fluorescence distributed along the chromosome, and good DAPI chromosome banding.
Good DAPI banding is critical for chromosome identification. If the
chromosomes are overdenatured or overdigested, hybridization may be
excellent, but the chromosomes may be unrecognizable. Therefore, the
experiment should balance between good hybridization and easy identification of chromosomes.
Excellent blocking of repeat sequences in chromosomes by CoT-1 DNA
is crucial (I 1). The goal of a CGH experiment is to measure a hybridization
that reflects the copy number of unique sequences in the test and reference
DNAs. If repeat sequences on the metaphase chromosomes are not well