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RENATE ULMER
2. Prepare the humidified hybridization chamber: an airtight container
with a piece of damp blotting paper.
3. Allow the probes to warm to room temperature for easier pipetting.
4. Spin the tubes briefly in a microcentrifuge to bring the contents to the
bottom of the tube.
5. Apply 5-7 f..ll (depending on number of nuclei on the slide) of CEP 181
X/Y probe mix to one hybridization area and 5-7 f..ll of LSI 13121 probe
mix to the other hybridization area on the slide(s). Immediately, place
a 15x 15 mm (for 5 f..ll probe) or a 20x20 mm (for 7 f..ll probe) coverslip
over the probe solution and allow the solution to spread evenly under
the coverslip. Air bubbles should be avoided.
6. Seal coverslip with rubber cement.
7. Place the slide(s) on the 74°C hot plate inside the water-bath (or take
slide-warmer with exactly this temperature) for 5-7 min (optimal time
must be tested).
8. Examine the seal of rubber-cement around the coverslips.
9. Place the slides into the hybridization chamber and incubate at 37°C
overnight.
Post-hybridization washes
1. Prewarm the 1xSSC (and OAxSSC for more stringent washing) solution by placing the Coplin jar in the 74°C water bath until the solution
has reached 73± 1°C.
2. Remove rubber cement seal and coverslips from the slides with forceps. Place the slides in the Coplin jar containing the 1x SSC at
73±I°C and incubate for 2 min.
3. Remove the slides from the wash bath and place in a jar with 2x SSCI
NP40 at RT for 2 min.
4. During this time prepare the DAPI-counterstain solution: per slide mix
1 ml of the 2xSSC/NP 40 + 1 f..ll DAPI stock solution.
5. Add 1 ml of the counterstain onto each slide and leave for 4 min in the
dark.
RENATE ULMER
2. Prepare the humidified hybridization chamber: an airtight container
with a piece of damp blotting paper.
3. Allow the probes to warm to room temperature for easier pipetting.
4. Spin the tubes briefly in a microcentrifuge to bring the contents to the
bottom of the tube.
5. Apply 5-7 f..ll (depending on number of nuclei on the slide) of CEP 181
X/Y probe mix to one hybridization area and 5-7 f..ll of LSI 13121 probe
mix to the other hybridization area on the slide(s). Immediately, place
a 15x 15 mm (for 5 f..ll probe) or a 20x20 mm (for 7 f..ll probe) coverslip
over the probe solution and allow the solution to spread evenly under
the coverslip. Air bubbles should be avoided.
6. Seal coverslip with rubber cement.
7. Place the slide(s) on the 74°C hot plate inside the water-bath (or take
slide-warmer with exactly this temperature) for 5-7 min (optimal time
must be tested).
8. Examine the seal of rubber-cement around the coverslips.
9. Place the slides into the hybridization chamber and incubate at 37°C
overnight.
Post-hybridization washes
1. Prewarm the 1xSSC (and OAxSSC for more stringent washing) solution by placing the Coplin jar in the 74°C water bath until the solution
has reached 73± 1°C.
2. Remove rubber cement seal and coverslips from the slides with forceps. Place the slides in the Coplin jar containing the 1x SSC at
73±I°C and incubate for 2 min.
3. Remove the slides from the wash bath and place in a jar with 2x SSCI
NP40 at RT for 2 min.
4. During this time prepare the DAPI-counterstain solution: per slide mix
1 ml of the 2xSSC/NP 40 + 1 f..ll DAPI stock solution.
5. Add 1 ml of the counterstain onto each slide and leave for 4 min in the
dark.