258
WILLIAM M. STRAUSS
that this treatment is particularly valuable in interphase FISH, resulting
in cleaner and stronger signals in telomeres or centromeres. Alternatively, slides can be treated 1 mg/ml ofpepsin pH 2.0 for 10 min at 37°C.
This step can, however, be omitted for PNA-FISH on telomeres, centromeres, or trinucleotide repeats (Chen et al. 1999).
Denature
the chromosomes
PNA-DNA
hybridization
Post-hybridization
washes
5. After washing in 1xPBS at 37°C for 5 min, slides are fixed in 4%
formaldehyde in 1x PBS, pH 7.2 at 37°C for 1.5 min for human chromosomes and 2 min for mouse chromosomes. After fixation, the slides
are immediately dehydrated with a cold ethanol series and air dried
for at least 10 min.
6. A total of 0.1 nmol ofPNA is needed for 10 to 15 slides. First, 0.1 nmol
of PNA is dissolved in 200 III of pre-warmed 1x hybridization mix containing 70% deionized formamide, 500 (50-1000) nM of fluorochromelabeled PNA probe, 1% (w/v) blocking reagent in 10 mM Tris pH 7.2
(no Na+). PNA hybridization mix is incubated at 37°C or 50°C in the
dark for at least 15 min. 15-20111 ofPNA (500 nM) is transferred to each
slide. A coverslip of 20x20 mm or 20x30 mm is applied and sealed
with rubber cement. The DNA on the slide is denatured again at
80°C for 3 min and then hybridized in a dark, wet chamber at 37°e
(20-40°C) for 2 h (0.5-24).
7. From this step, excessive light should be avoided to prevent fluorescence breaching. After hybridization, slide preparations are washed
three times with 70% formamide/lO mM Tris pH 7.2 for 10 min
each and with 50 mM Trisl150 mM NaCl/0.05% Tween-20 pH 7.5
for 5 min each. The slides are then dehydrated in an ethanol series
and air dried in the dark. Chromosomes are counterstained with either
O.IIlg/ml of4,6-diamidino-2-phenylindole-dehydrocWoride (DAPI) in
antifade for rhodamine- and fluorescein-labeled probes or 0.6 Ilg/ml of
propidium iodide (PI) for fluorescein-labeled probes. It is recommended to analyze slides afterwards. Fluorescent signals can, however,
be well retained at -20
o e for a few weeks.
WILLIAM M. STRAUSS
that this treatment is particularly valuable in interphase FISH, resulting
in cleaner and stronger signals in telomeres or centromeres. Alternatively, slides can be treated 1 mg/ml ofpepsin pH 2.0 for 10 min at 37°C.
This step can, however, be omitted for PNA-FISH on telomeres, centromeres, or trinucleotide repeats (Chen et al. 1999).
Denature
the chromosomes
PNA-DNA
hybridization
Post-hybridization
washes
5. After washing in 1xPBS at 37°C for 5 min, slides are fixed in 4%
formaldehyde in 1x PBS, pH 7.2 at 37°C for 1.5 min for human chromosomes and 2 min for mouse chromosomes. After fixation, the slides
are immediately dehydrated with a cold ethanol series and air dried
for at least 10 min.
6. A total of 0.1 nmol ofPNA is needed for 10 to 15 slides. First, 0.1 nmol
of PNA is dissolved in 200 III of pre-warmed 1x hybridization mix containing 70% deionized formamide, 500 (50-1000) nM of fluorochromelabeled PNA probe, 1% (w/v) blocking reagent in 10 mM Tris pH 7.2
(no Na+). PNA hybridization mix is incubated at 37°C or 50°C in the
dark for at least 15 min. 15-20111 ofPNA (500 nM) is transferred to each
slide. A coverslip of 20x20 mm or 20x30 mm is applied and sealed
with rubber cement. The DNA on the slide is denatured again at
80°C for 3 min and then hybridized in a dark, wet chamber at 37°e
(20-40°C) for 2 h (0.5-24).
7. From this step, excessive light should be avoided to prevent fluorescence breaching. After hybridization, slide preparations are washed
three times with 70% formamide/lO mM Tris pH 7.2 for 10 min
each and with 50 mM Trisl150 mM NaCl/0.05% Tween-20 pH 7.5
for 5 min each. The slides are then dehydrated in an ethanol series
and air dried in the dark. Chromosomes are counterstained with either
O.IIlg/ml of4,6-diamidino-2-phenylindole-dehydrocWoride (DAPI) in
antifade for rhodamine- and fluorescein-labeled probes or 0.6 Ilg/ml of
propidium iodide (PI) for fluorescein-labeled probes. It is recommended to analyze slides afterwards. Fluorescent signals can, however,
be well retained at -20
o e for a few weeks.