Preparation and storage of metaphase chromosomes
Metaphase chromosomes of the species under investigation are preferably
obtained from primary fibroblast cultures or cultured peripheral blood
lymphocytes. Chromosomes are spread according to standard methanol/acetic acid (3/1) fixation protocols (Islam and Levan 1987, Fronicke
and Scherthan 1997).
1. Grow fibroblasts to half confluency, arrest them at metaphase by colcemid treatment and swell cells for 30 min in 0.0375 M KCl.
2. Fix the cell pellet more than five times with always freshly prepared ice
cold fixative.
3. Dissolve pellet in 0.5 ml fixative and drop -50 III of cell suspension on
an ethanol-cleaned slide, tilt and drain excess fixative.
4. Place slide over a water bath at 2':60°e. Let slides sit until the upper side
appears dry.
5. Remove preparations and inspect them by phase contrast microscopy
on low power. Cytoplasm is evident as dark rings around metaphase
plates and nuclei. Repeat fixation until cytoplasm is absent. In phase
contrast chromosomes should have an evenly dark appearance and
metaphase plates should be closely spaced.
6. Allow dried slides to sit at RT for up to 2 h.
7. Slides are sealed in a plastic container and stored at -20°e.
Pretreatment and denaturation of chromosome preparations
Prior to in situ hybridization, fresh or thawed chromosome preparations
are placed for 50 min on a heating block or hot plate at 90°C to harden
chromosomal chromatin, which limits swelling of the chromosomes during denaturation and extended hybridization time.
1. Remove slides with forceps and allow to cool to room temperature.
2. Cover preparations with 100 III 70% formamide/30% 2xSSe, pH 7.0.
3. Mount a 22x60 mm coverslip and incubate for 2 min on a hot plate or
heating block at 70
0 e to denature chromosomal chromatin.
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Metaphase chromosomes of the species under investigation are preferably
obtained from primary fibroblast cultures or cultured peripheral blood
lymphocytes. Chromosomes are spread according to standard methanol/acetic acid (3/1) fixation protocols (Islam and Levan 1987, Fronicke
and Scherthan 1997).
1. Grow fibroblasts to half confluency, arrest them at metaphase by colcemid treatment and swell cells for 30 min in 0.0375 M KCl.
2. Fix the cell pellet more than five times with always freshly prepared ice
cold fixative.
3. Dissolve pellet in 0.5 ml fixative and drop -50 III of cell suspension on
an ethanol-cleaned slide, tilt and drain excess fixative.
4. Place slide over a water bath at 2':60°e. Let slides sit until the upper side
appears dry.
5. Remove preparations and inspect them by phase contrast microscopy
on low power. Cytoplasm is evident as dark rings around metaphase
plates and nuclei. Repeat fixation until cytoplasm is absent. In phase
contrast chromosomes should have an evenly dark appearance and
metaphase plates should be closely spaced.
6. Allow dried slides to sit at RT for up to 2 h.
7. Slides are sealed in a plastic container and stored at -20°e.
Pretreatment and denaturation of chromosome preparations
Prior to in situ hybridization, fresh or thawed chromosome preparations
are placed for 50 min on a heating block or hot plate at 90°C to harden
chromosomal chromatin, which limits swelling of the chromosomes during denaturation and extended hybridization time.
1. Remove slides with forceps and allow to cool to room temperature.
2. Cover preparations with 100 III 70% formamide/30% 2xSSe, pH 7.0.
3. Mount a 22x60 mm coverslip and incubate for 2 min on a hot plate or
heating block at 70
0 e to denature chromosomal chromatin.
25 Zoo-FISH
315