352
MONICA MUSIANI et al.
tively, with a covalently attached signaling group that is usually AP or
HRP. Digoxigenin labeled probes have proven to be very sensitive and
specific for the detection of viral genomes, and nowadays are the most
used probes.
Hybridization
mixture
- 50% deionized formamide
- 10% dextran sulfate
- 250 Jlg/ml of carrier calf thymus DNA
- 2 ng/JlI of the labeled probe in 2xSSC buffer (0.3 M NaCI, 0.03 M sodium citrate, pH 7.0)
Procedure
Several protocols for the hybridization reaction have been described with
minor modifications, generally involving a cell pretreatment followed by
the hybridization reaction with the labeled probe, followed by chemiluminescent detection.
Pre-treatment of cells/sections
1. Hydrate cells/sections briefly in PBS.
2. Place cells/sections in 0.02 N HCI for 10 min.
3. Wash three times with PBS.
4. Treat cells/sections with 0.01% Triton X-lOa in PBS for 2 min.
5. Wash three times with PBS.
6. Treat with pronase (0.5 mg/ml) in 0.05 M Tris HCI buffer, pH 7.6, containing 5 mM EDTA for 5 min.
7. Wash twice with PBS containing 2 mg/ml of glycine.
8. Postfix with 4% paraformaldehyde in PBS for 5 min.
9. Wash twice with PBS containing 2 mg/ml of glycine.
10. Dehydrate cells/sections by ethanol washes (30%,60%,80%,95% and
100%).
MONICA MUSIANI et al.
tively, with a covalently attached signaling group that is usually AP or
HRP. Digoxigenin labeled probes have proven to be very sensitive and
specific for the detection of viral genomes, and nowadays are the most
used probes.
Hybridization
mixture
- 50% deionized formamide
- 10% dextran sulfate
- 250 Jlg/ml of carrier calf thymus DNA
- 2 ng/JlI of the labeled probe in 2xSSC buffer (0.3 M NaCI, 0.03 M sodium citrate, pH 7.0)
Procedure
Several protocols for the hybridization reaction have been described with
minor modifications, generally involving a cell pretreatment followed by
the hybridization reaction with the labeled probe, followed by chemiluminescent detection.
Pre-treatment of cells/sections
1. Hydrate cells/sections briefly in PBS.
2. Place cells/sections in 0.02 N HCI for 10 min.
3. Wash three times with PBS.
4. Treat cells/sections with 0.01% Triton X-lOa in PBS for 2 min.
5. Wash three times with PBS.
6. Treat with pronase (0.5 mg/ml) in 0.05 M Tris HCI buffer, pH 7.6, containing 5 mM EDTA for 5 min.
7. Wash twice with PBS containing 2 mg/ml of glycine.
8. Postfix with 4% paraformaldehyde in PBS for 5 min.
9. Wash twice with PBS containing 2 mg/ml of glycine.
10. Dehydrate cells/sections by ethanol washes (30%,60%,80%,95% and
100%).