340
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
nals (due to the small amount of fluorochromes deposited at the target
region[s)), hence analysis of such FISH experiments requires image recording with a charge-coupled device camera and computer-based image
analysis system.
Procedure
1. Label 1 ~g of probe DNA by the method of your choice (see above).
Remove 1/ 20 of the reaction volume to perform a minigel analysis.
2. To the labeling reaction, add 1/ 10 volume of300mM EDTA, 1 ~l carrier
DNA and precipitate the DNA by adding 3 volumes of ethanol.
3. 4. Incubate for 30 min at -20°C, centrifuge for 30 min at 12,000 g and
resuspend pellet in hybridization solution to result in a final probe
DNA concentration of 30-50 ng/~l.
4. Place tube for 30 min at 60°C to completely dissolve the pellet in the
hybridization solution.
5. Denature hybridization solution for 3 min at 95°C, coolon ice.
6. On the preparations, determine the region with highest cell density on
the slide and mark it from underneath with a diamond pen.
7. Denature preparations by applying 100 ~l of 70% formamid/2 x SSC
under a large coverslip and place slide for 5 min at 74°C. Wash
down coverslip and denaturation solution with a jet of ice cold water
and air dry.
8. Add required amount ofhybridization solution (l ~l for a round coverslip of 10 mm diameter is sufficient) to the marked region, add cover
slip and seal with rubber cement. Slides are placed in a dry box and
renaturation is allowed to proceed at 37°C for 2 days in a moist incubator (Scherthan et al. 1992).
9. After in situ hybridization, rubber cement is removed with forceps and
coverslips are floated off in a Coplin jar containing 0.05xSSC.
10. Perform 3x 5 min stringency washes in 0.05 x SSC at 42°C to remove
excess and unspecifically bound probe molecules.
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
nals (due to the small amount of fluorochromes deposited at the target
region[s)), hence analysis of such FISH experiments requires image recording with a charge-coupled device camera and computer-based image
analysis system.
Procedure
1. Label 1 ~g of probe DNA by the method of your choice (see above).
Remove 1/ 20 of the reaction volume to perform a minigel analysis.
2. To the labeling reaction, add 1/ 10 volume of300mM EDTA, 1 ~l carrier
DNA and precipitate the DNA by adding 3 volumes of ethanol.
3. 4. Incubate for 30 min at -20°C, centrifuge for 30 min at 12,000 g and
resuspend pellet in hybridization solution to result in a final probe
DNA concentration of 30-50 ng/~l.
4. Place tube for 30 min at 60°C to completely dissolve the pellet in the
hybridization solution.
5. Denature hybridization solution for 3 min at 95°C, coolon ice.
6. On the preparations, determine the region with highest cell density on
the slide and mark it from underneath with a diamond pen.
7. Denature preparations by applying 100 ~l of 70% formamid/2 x SSC
under a large coverslip and place slide for 5 min at 74°C. Wash
down coverslip and denaturation solution with a jet of ice cold water
and air dry.
8. Add required amount ofhybridization solution (l ~l for a round coverslip of 10 mm diameter is sufficient) to the marked region, add cover
slip and seal with rubber cement. Slides are placed in a dry box and
renaturation is allowed to proceed at 37°C for 2 days in a moist incubator (Scherthan et al. 1992).
9. After in situ hybridization, rubber cement is removed with forceps and
coverslips are floated off in a Coplin jar containing 0.05xSSC.
10. Perform 3x 5 min stringency washes in 0.05 x SSC at 42°C to remove
excess and unspecifically bound probe molecules.