12 Chromosome Analysis by Fluorescence in situ Hybridization
179
4. Transfer the slides to a Coplin jar containing prewarmed (60°C) wash
solution B, and wash for 5 minutes. Changesolution twice, each time
washing for 5 minutes.
5. Remove the slides from the jar, drain, and apply 200 !J.l ofblocking solution. Cover with a 22x40mm coverslip, place in a humidified chamber
and incubate at 37°C for at least 30 minutes.
6. Remove the coverslip from each slide, drain excess fluid, and add 200 !J.l
of detection solution containing the fluorochrome-conjugated reporter-detecting reagent (e.g. 5!J.g/ml fluorescein-conjugated avidin or 6
!J.g/ml rhodamine conjugated anti-digoxigenin). Incubate in a humidified chamber at 37°C for 30 minutes. All subsequent steps should be
carried out in light-protected Coplinjars (e.g. wrapped with aluminium
foil).
7. Remove the coverslip, transfer the slides into wash solution C, and wash
3 times for 5 minutes at 42°C (shaking water bath).
8. Place slides in a Coplin jar containing counterstaining solution ( e.g. 2x
SSC, 200nglml DAPI) and agitate at room temperature for 20 minutes.
9. Transferslides into a jar containing wash solution D. Incubate for 1 to 2
minutes at room temperature.
10. Remove each slide from the Coplin jar, add 20 to 30 !J.l of antifade solution and cover with a 22x40 mm coverslip. Place slides in suitable boxes,
which should be kept at 4°C for long-term storage.
Subprotocol 7
Chromosomal in situ Suppression (CISS) Hybridization
This protocol is used when the probes contain IRS sequences, as is the case
for almost all genomic DNA probes. For the Suppression of non-specific
signals arising from theserepetitive segments, the DNA probe is pre-mixed
with an excess of unlabeled competitor DNA and an additional pre-annealing step is performed afterprobe denaturation (Lichteret al. 1988; Pinkelet
al1988). Again, a protocol is provided for a 10 !J.l hybridization volume.
Probes as small as a few kb as well as probes targeting many megabases
can be used for CISS hybridization. Therefore the concentration of the individual probe in the hybridization volume must be adjusted according to
the complexitiy of the target region. The following list provides the quantities of probes required for a 10 !J.l reaction:
179
4. Transfer the slides to a Coplin jar containing prewarmed (60°C) wash
solution B, and wash for 5 minutes. Changesolution twice, each time
washing for 5 minutes.
5. Remove the slides from the jar, drain, and apply 200 !J.l ofblocking solution. Cover with a 22x40mm coverslip, place in a humidified chamber
and incubate at 37°C for at least 30 minutes.
6. Remove the coverslip from each slide, drain excess fluid, and add 200 !J.l
of detection solution containing the fluorochrome-conjugated reporter-detecting reagent (e.g. 5!J.g/ml fluorescein-conjugated avidin or 6
!J.g/ml rhodamine conjugated anti-digoxigenin). Incubate in a humidified chamber at 37°C for 30 minutes. All subsequent steps should be
carried out in light-protected Coplinjars (e.g. wrapped with aluminium
foil).
7. Remove the coverslip, transfer the slides into wash solution C, and wash
3 times for 5 minutes at 42°C (shaking water bath).
8. Place slides in a Coplin jar containing counterstaining solution ( e.g. 2x
SSC, 200nglml DAPI) and agitate at room temperature for 20 minutes.
9. Transferslides into a jar containing wash solution D. Incubate for 1 to 2
minutes at room temperature.
10. Remove each slide from the Coplin jar, add 20 to 30 !J.l of antifade solution and cover with a 22x40 mm coverslip. Place slides in suitable boxes,
which should be kept at 4°C for long-term storage.
Subprotocol 7
Chromosomal in situ Suppression (CISS) Hybridization
This protocol is used when the probes contain IRS sequences, as is the case
for almost all genomic DNA probes. For the Suppression of non-specific
signals arising from theserepetitive segments, the DNA probe is pre-mixed
with an excess of unlabeled competitor DNA and an additional pre-annealing step is performed afterprobe denaturation (Lichteret al. 1988; Pinkelet
al1988). Again, a protocol is provided for a 10 !J.l hybridization volume.
Probes as small as a few kb as well as probes targeting many megabases
can be used for CISS hybridization. Therefore the concentration of the individual probe in the hybridization volume must be adjusted according to
the complexitiy of the target region. The following list provides the quantities of probes required for a 10 !J.l reaction: