24 Fluorescence in Situ Hybridisation of a Complete Set of 41 Telomere Specific Probes
305
7. Carefully lower the slide over the Multiprobe so that the drops of hybridisation fluid make contact with the slide. Apply gentle, even pressure to ensure that the hybridisation fluid is spread to the edges of each
of the raised areas on the Multiprobe.
8. Lift the slide/Multiprobe carefully and invert so that the slide is under
the Multiprobe.
9. Incubate at 37°C for 10 min.
10. Transfer the slide/Multiprobe device to the 75°C hotplate taking particular care to hold it level (ensure the sample slide is in good contact
with the hotplate). Incubate at 75°C (+/-lOC) for 2 min.
11. Place slide/Multiprobe device in the Chromoprobe Multiprobe Hybridisation Chamber, replace, but do not seal, the lid and float the chamber in an uncovered 37°C water bath overnight. An incubator should
not be used for the hybridisation procedure, as the multiprobe system
is an open one and the correct levels of humidity are vital for good
hybridisation. Damp tissues must not be used inside the chamber.
Post hybridisation washes
1. Remove the Multiprobe device carefully from the sample slide and incubate the slide in OAxSSC (pH 7.0) at noc for 2 min.
2. Remove the slide and incubate in 2xSSC, 0.05% Tween 20 for 30 s at
room temperature.
3. Drain off excess solution by placing the slide on its edge on a tissue for
around 20 s. Do not allow to dry.
4. Add one 20 JlI drop ofDAPI-antifade solution to both ends of the slide
and apply a large (24x60 mm) coverslip slowly to avoid the formation
of air bubbles.
8 7 [!][I)[!][l]1II1i)
16 15 14 13 12 11 ~III
xv 22 21 20 19 [!!J[!2] Fig. 3. Location of telomeric probes
on Multiprobe bosses
305
7. Carefully lower the slide over the Multiprobe so that the drops of hybridisation fluid make contact with the slide. Apply gentle, even pressure to ensure that the hybridisation fluid is spread to the edges of each
of the raised areas on the Multiprobe.
8. Lift the slide/Multiprobe carefully and invert so that the slide is under
the Multiprobe.
9. Incubate at 37°C for 10 min.
10. Transfer the slide/Multiprobe device to the 75°C hotplate taking particular care to hold it level (ensure the sample slide is in good contact
with the hotplate). Incubate at 75°C (+/-lOC) for 2 min.
11. Place slide/Multiprobe device in the Chromoprobe Multiprobe Hybridisation Chamber, replace, but do not seal, the lid and float the chamber in an uncovered 37°C water bath overnight. An incubator should
not be used for the hybridisation procedure, as the multiprobe system
is an open one and the correct levels of humidity are vital for good
hybridisation. Damp tissues must not be used inside the chamber.
Post hybridisation washes
1. Remove the Multiprobe device carefully from the sample slide and incubate the slide in OAxSSC (pH 7.0) at noc for 2 min.
2. Remove the slide and incubate in 2xSSC, 0.05% Tween 20 for 30 s at
room temperature.
3. Drain off excess solution by placing the slide on its edge on a tissue for
around 20 s. Do not allow to dry.
4. Add one 20 JlI drop ofDAPI-antifade solution to both ends of the slide
and apply a large (24x60 mm) coverslip slowly to avoid the formation
of air bubbles.
8 7 [!][I)[!][l]1II1i)
16 15 14 13 12 11 ~III
xv 22 21 20 19 [!!J[!2] Fig. 3. Location of telomeric probes
on Multiprobe bosses