11 Specimen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
159
• Denaturation solution: 70% deionized formamide/2x SSC/50 mM sodium phosphate, adjusted to pH 7
• Hybridization solution: 50% deionized formamide/2x SSC/10% dextran
sulfate
• Rubber cement
Procedure
1. Place sample of solid tumor (0.5 - 1g, if available) in an ice-cold petri
dish and mince with a scalpel.
2. Suspend cells in 70% ethanol ( -20°C). If necessary, further homogenize
tissue in a Dounc homogenizer. The material can be stored in 70% ethanol at -20°C.
3. Suspend tumor cells to a concentration of approximately 5x1 0 5 cells/ml.
Place 10 J..Ll of cell suspension on poly-L-lysine coated slides. Mark area
containing cells using a diamond pen on the opposite side of the slide.
4. Air dry for 15 min.
5. Add200 J..Ll ofdigestion solution andincubate forabout 15 min (see note
1 below). It may be necessaryto modifythe incubation time empirically.
6. Briefly wash slides 5 times in water, then 5 times in PBS.
7. Fix the cells in 1 o/o formaldehyde, dissolved in 0.1 M phosphate buffer
for 10 min at room temperature.
8. Briefly wash slides 5 times in water and 5 times in PBS.
9. Dehydrate slides in 70%,90% and 100% ethanol for 5 min each and air
dry.
10. Denature slides in denaturation solution at 70°C for 2 min in a Coplin
jar.
11. Immediatedly dehydrate in ice-cold 70%, 90% and 100% ethanol ( 5 min
each) and air dry.
12. Add hybridization solution containing denaturated probe (see note 2
below) and hybridize overnight at 37°C in a humidified chamber. For
detection of hybridized probes see Chap. 12).
Preparation of
single cell
suspension
159
• Denaturation solution: 70% deionized formamide/2x SSC/50 mM sodium phosphate, adjusted to pH 7
• Hybridization solution: 50% deionized formamide/2x SSC/10% dextran
sulfate
• Rubber cement
Procedure
1. Place sample of solid tumor (0.5 - 1g, if available) in an ice-cold petri
dish and mince with a scalpel.
2. Suspend cells in 70% ethanol ( -20°C). If necessary, further homogenize
tissue in a Dounc homogenizer. The material can be stored in 70% ethanol at -20°C.
3. Suspend tumor cells to a concentration of approximately 5x1 0 5 cells/ml.
Place 10 J..Ll of cell suspension on poly-L-lysine coated slides. Mark area
containing cells using a diamond pen on the opposite side of the slide.
4. Air dry for 15 min.
5. Add200 J..Ll ofdigestion solution andincubate forabout 15 min (see note
1 below). It may be necessaryto modifythe incubation time empirically.
6. Briefly wash slides 5 times in water, then 5 times in PBS.
7. Fix the cells in 1 o/o formaldehyde, dissolved in 0.1 M phosphate buffer
for 10 min at room temperature.
8. Briefly wash slides 5 times in water and 5 times in PBS.
9. Dehydrate slides in 70%,90% and 100% ethanol for 5 min each and air
dry.
10. Denature slides in denaturation solution at 70°C for 2 min in a Coplin
jar.
11. Immediatedly dehydrate in ice-cold 70%, 90% and 100% ethanol ( 5 min
each) and air dry.
12. Add hybridization solution containing denaturated probe (see note 2
below) and hybridize overnight at 37°C in a humidified chamber. For
detection of hybridized probes see Chap. 12).
Preparation of
single cell
suspension