426
ANGELA NIETZEL et al.
marker chromosomes with no - or nearly no - euchromatin and small
amounts of available chromosome suspension (see Fig. 1A).
Outline
1. FISH is performed on metaphase spreads from a lymphocyte suspension, amniocytic suspension or bone marrow; slides are treated with
RNase and pepsin prior to denaturation.
2. Denaturation of the slides is performed for about 2-3 min at 75°C. The
probes are denatured for 5 min and hybridized on the slides for 1 night
at 37°C in a humid chamber. After a postwashing series, detection of
the biotinylated probe is performed with a Cy5-avidin system. After
counterstaining with DAPI and addition of antifade solution, slides
can be evaluated on a six-filter fluorescence microscope equipped
with a CCD-camera and a computer with image-analyzing system.
Procedure
Slide pretreatment
1. Slides with the chromosome suspension are incubated in 2x SSC for
5 min at RT (in a 100-ml Coplin jar on a shaker).
2. Remove slides from the Coplin jar, add 100 III of RNase solution/slide
and cover with a 24xSO mm coverslip (RNase solution: per slide 100 III
2xSSC plus 1 III of RNase stock (=5 1lg/1l1)}.
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment l).
4. Put slides back into the Coplin jar with 100 ml2 x SSC (RT) and remove
the coverslips with forceps. Leave slides in 2x SSC solution for 3 min
with gentle agitation.
5. Discard 2xSSC and replace it with 100 ml1xPBS (RT) for 5 min (shaker).
6. Replace 1x PBS with 100 ml prewarmed pepsin-buffer (37°C) and incubate the slides for 10 min at 37°C, without agitation (see comment l).
[Pepsin-buffer: Add 1 ml of 1 M HCI to 99 ml of distilled water and
incubate at 37°C for about 20 min; then add 50 III of the pepsin stock
ANGELA NIETZEL et al.
marker chromosomes with no - or nearly no - euchromatin and small
amounts of available chromosome suspension (see Fig. 1A).
Outline
1. FISH is performed on metaphase spreads from a lymphocyte suspension, amniocytic suspension or bone marrow; slides are treated with
RNase and pepsin prior to denaturation.
2. Denaturation of the slides is performed for about 2-3 min at 75°C. The
probes are denatured for 5 min and hybridized on the slides for 1 night
at 37°C in a humid chamber. After a postwashing series, detection of
the biotinylated probe is performed with a Cy5-avidin system. After
counterstaining with DAPI and addition of antifade solution, slides
can be evaluated on a six-filter fluorescence microscope equipped
with a CCD-camera and a computer with image-analyzing system.
Procedure
Slide pretreatment
1. Slides with the chromosome suspension are incubated in 2x SSC for
5 min at RT (in a 100-ml Coplin jar on a shaker).
2. Remove slides from the Coplin jar, add 100 III of RNase solution/slide
and cover with a 24xSO mm coverslip (RNase solution: per slide 100 III
2xSSC plus 1 III of RNase stock (=5 1lg/1l1)}.
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment l).
4. Put slides back into the Coplin jar with 100 ml2 x SSC (RT) and remove
the coverslips with forceps. Leave slides in 2x SSC solution for 3 min
with gentle agitation.
5. Discard 2xSSC and replace it with 100 ml1xPBS (RT) for 5 min (shaker).
6. Replace 1x PBS with 100 ml prewarmed pepsin-buffer (37°C) and incubate the slides for 10 min at 37°C, without agitation (see comment l).
[Pepsin-buffer: Add 1 ml of 1 M HCI to 99 ml of distilled water and
incubate at 37°C for about 20 min; then add 50 III of the pepsin stock