- Formamide, research grade (Merck). A small quantity of deionized formamide is required for the hybridization solution. Small aliquots are
prepared by filling the tip of a 1.5 ml microfuge tube with ion exchange
resin (20-50 mesh; Biorad). Add 1 ml of formamide, mix and store at
-20°e.
Note: Formamide is a harmful chemical and should be handled with
care.
- 70% formamide/30% 2xSSC, pH 7.0. This denaturation solution can
be stored for several weeks at 4°e.
- Cotl DNA from human and other species may be obtained from a commercial supplier. In case it is not available, Cotl DNA may be prepared
according to published protocols (Kappler et al. 1998).
- Hybridization solution is composed of: 50% deionized formamide,
10% 20xSSC, 20% of a 50% (w/v) dextran sulfate (Pharmacia) solution,
10% 50 mM sodium phosphate pH 7.0, human Cotl DNA at a final
cone. of2 /lg//ll, probe DNA at 300 ng//ll. Use deionized water to adjust
volume.
- Tween-20 (Sigma)
- BT buffer (0.15 M NaHC0 3 , 0.1% Tween-20, pH 8.3). After dissolution
of NaHC0 3 , pH will be in the appropriate range. Make up fresh BT for
each experiment.
- BSA (fraction V, Serva)
- Avidin-FITC (e.g., ExtrAvidin-fluorescein, Sigma)
- Biotinylated goat-anti-avidin antiserum (Vector Labs)
- Propidium iodide (PI, Sigma). Dissolve at 1 mg/ml in sterile water,
store stock at -20°e.
- DAPI (4,6-Diamidine-2-phenylindole dihydrochloride) (Sigma). Stock
solution: 1 mg/ml in sterile water, store at -20°e.
- Antifade solution: Vectashield (Vector Labs) is recommended as a
mounting medium for fluorescence microscopy, since it efficiently reduces fading of fluorochromes during microscopic analysis (Florijn et
al. 1995). For counter staining of DNA, fluorescent dyes are added to
the antifade solution in the following concentrations: DAPI 0.5 /lg/ml
and/or PI 1 /lg/ml.
25 Zoo-FISH
313
prepared by filling the tip of a 1.5 ml microfuge tube with ion exchange
resin (20-50 mesh; Biorad). Add 1 ml of formamide, mix and store at
-20°e.
Note: Formamide is a harmful chemical and should be handled with
care.
- 70% formamide/30% 2xSSC, pH 7.0. This denaturation solution can
be stored for several weeks at 4°e.
- Cotl DNA from human and other species may be obtained from a commercial supplier. In case it is not available, Cotl DNA may be prepared
according to published protocols (Kappler et al. 1998).
- Hybridization solution is composed of: 50% deionized formamide,
10% 20xSSC, 20% of a 50% (w/v) dextran sulfate (Pharmacia) solution,
10% 50 mM sodium phosphate pH 7.0, human Cotl DNA at a final
cone. of2 /lg//ll, probe DNA at 300 ng//ll. Use deionized water to adjust
volume.
- Tween-20 (Sigma)
- BT buffer (0.15 M NaHC0 3 , 0.1% Tween-20, pH 8.3). After dissolution
of NaHC0 3 , pH will be in the appropriate range. Make up fresh BT for
each experiment.
- BSA (fraction V, Serva)
- Avidin-FITC (e.g., ExtrAvidin-fluorescein, Sigma)
- Biotinylated goat-anti-avidin antiserum (Vector Labs)
- Propidium iodide (PI, Sigma). Dissolve at 1 mg/ml in sterile water,
store stock at -20°e.
- DAPI (4,6-Diamidine-2-phenylindole dihydrochloride) (Sigma). Stock
solution: 1 mg/ml in sterile water, store at -20°e.
- Antifade solution: Vectashield (Vector Labs) is recommended as a
mounting medium for fluorescence microscopy, since it efficiently reduces fading of fluorochromes during microscopic analysis (Florijn et
al. 1995). For counter staining of DNA, fluorescent dyes are added to
the antifade solution in the following concentrations: DAPI 0.5 /lg/ml
and/or PI 1 /lg/ml.
25 Zoo-FISH
313