20 Strand-Specific Fluorescence in Situ Hybridization: CO-FISH and COD-FISH
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3. Add Colcemid to collect cells at metaphase. We use 0.1-0.2 flg/ml final
concentration for the last 2-4 h of culture for human fibroblast cultures.
4. Harvest the cell culture using standard methods. For example, for metaphase chromosome preparation of human fibroblast cultures, we use
a 15 min hypotonic treatment in 75 mM KCI solution followed by fixation in 3:1 methanol-acetic acid. The cell suspension is dropped onto
clean glass microscope slides and allowed to air dry. Slides are best if
aged at least 24 h before use.
Preparation of single-stranded DNA
1. Stain the slides with a solution of 0.5 flg/ml Hoechst 33258 in 2 x SSC for
15 min at room temperature.
2. Rinse briefly in distilled water and air dry.
3. Place 2-3 drops of 2 x SSC on each slide and cover with a 22 x 50 mm #1
grade glass coverslip.
4. Expose to long-wave UV light for 30 min.
5. Remove the coverslip, rinse briefly in distilled water and air dry.
6. Place 50 fll of exonuclease III solution (3 Uilli in buffer supplied by
manufacturer) onto the slide and cover with a 22x50 mm glass coverslip. We have used exonuclease III from a variety of sources with equal
success.
7. Incubate at room temperature for 5 min. Some cell types or slides more
than a few weeks old may require longer times of exonuclease incubation.
8. Remove the coverslip, rinse briefly in distilled water and air dry.
Hybridization and detection of probe
No denaturation step is necessary because the DNA is single-stranded. The
hybridization mix, incubation and detection procedure should be the
same as for standard FISH with the same probe.
1. Prepare probe hybridization mix, place it on the slide and cover with a
coverslip.
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3. Add Colcemid to collect cells at metaphase. We use 0.1-0.2 flg/ml final
concentration for the last 2-4 h of culture for human fibroblast cultures.
4. Harvest the cell culture using standard methods. For example, for metaphase chromosome preparation of human fibroblast cultures, we use
a 15 min hypotonic treatment in 75 mM KCI solution followed by fixation in 3:1 methanol-acetic acid. The cell suspension is dropped onto
clean glass microscope slides and allowed to air dry. Slides are best if
aged at least 24 h before use.
Preparation of single-stranded DNA
1. Stain the slides with a solution of 0.5 flg/ml Hoechst 33258 in 2 x SSC for
15 min at room temperature.
2. Rinse briefly in distilled water and air dry.
3. Place 2-3 drops of 2 x SSC on each slide and cover with a 22 x 50 mm #1
grade glass coverslip.
4. Expose to long-wave UV light for 30 min.
5. Remove the coverslip, rinse briefly in distilled water and air dry.
6. Place 50 fll of exonuclease III solution (3 Uilli in buffer supplied by
manufacturer) onto the slide and cover with a 22x50 mm glass coverslip. We have used exonuclease III from a variety of sources with equal
success.
7. Incubate at room temperature for 5 min. Some cell types or slides more
than a few weeks old may require longer times of exonuclease incubation.
8. Remove the coverslip, rinse briefly in distilled water and air dry.
Hybridization and detection of probe
No denaturation step is necessary because the DNA is single-stranded. The
hybridization mix, incubation and detection procedure should be the
same as for standard FISH with the same probe.
1. Prepare probe hybridization mix, place it on the slide and cover with a
coverslip.