33 Simultaneous Multicolor-FISH and Immunocytochemical Analysis of Fresh Tumor Material
437
5. Place the slides for 10 min in a 1% acid-free formaldehyde/I x PBS/50
mM MgClz solution at RT.
6. Rinse the slides 1 min in 1x PBS at RT.
7. Incubate the slides 10 min in lxPBS at RT.
8. Dehydrate the slides through an ethanol series and air dry.
9. Apply 120 III of denaturation mixture on each slide.
10. Cover the solution with a 24 x 60 mm coverslip.
11. Denature 2 min at 80°C on a heating plate.
12. Remove coverslips and place the slides immediately in 70% ethanol at
-20°C for 5 min, then dehydrate in 80% and 95% ethanol at -20°C each
for 5 min and air dry.
13. Pre-warm the slides on a heating plate at 37°C.
1. Take the required amount of single copy probe DNA (e.g. 150 ng).
2. Add 50x human Cot! DNA.
3. Add 0.1 x volume 3 M sodium acetate pH 5.5.
4. Add 2.5 x volume 100% ethanol.
5. Mix, spin down and place it for 2 h at -20°C.
6. Spin down (30 min, 4°C, 14,000 rpm).
7. Remove supernatant and dry the pellet for 5 min in a speed-vac.
8. Resuspend the pellet in 20 III hybridization mixture and dissolve for 1 h
by gentle mixing.
9. Denature the DNA for 5 min at 80°C.
10. Place the DNA immediately on ice for 1 min and spin down.
11. Pre-anneal the DNA for 20 min at 37°C.
1. Denature 10 III of the a-DNA probe dissolved in hybridization mixture
(6 ng/Ill) for 10 min at 80°C, place it on ice for 1 min and warm it again
at 37°C.
2. Add the a-satellite DNA probe to the pre-annealed single copy probe,
mix well and spin down for a few seconds.
Probe preparation
and denaturation
At the same time
prepare the
a-satellite DNA
probe
437
5. Place the slides for 10 min in a 1% acid-free formaldehyde/I x PBS/50
mM MgClz solution at RT.
6. Rinse the slides 1 min in 1x PBS at RT.
7. Incubate the slides 10 min in lxPBS at RT.
8. Dehydrate the slides through an ethanol series and air dry.
9. Apply 120 III of denaturation mixture on each slide.
10. Cover the solution with a 24 x 60 mm coverslip.
11. Denature 2 min at 80°C on a heating plate.
12. Remove coverslips and place the slides immediately in 70% ethanol at
-20°C for 5 min, then dehydrate in 80% and 95% ethanol at -20°C each
for 5 min and air dry.
13. Pre-warm the slides on a heating plate at 37°C.
1. Take the required amount of single copy probe DNA (e.g. 150 ng).
2. Add 50x human Cot! DNA.
3. Add 0.1 x volume 3 M sodium acetate pH 5.5.
4. Add 2.5 x volume 100% ethanol.
5. Mix, spin down and place it for 2 h at -20°C.
6. Spin down (30 min, 4°C, 14,000 rpm).
7. Remove supernatant and dry the pellet for 5 min in a speed-vac.
8. Resuspend the pellet in 20 III hybridization mixture and dissolve for 1 h
by gentle mixing.
9. Denature the DNA for 5 min at 80°C.
10. Place the DNA immediately on ice for 1 min and spin down.
11. Pre-anneal the DNA for 20 min at 37°C.
1. Denature 10 III of the a-DNA probe dissolved in hybridization mixture
(6 ng/Ill) for 10 min at 80°C, place it on ice for 1 min and warm it again
at 37°C.
2. Add the a-satellite DNA probe to the pre-annealed single copy probe,
mix well and spin down for a few seconds.
Probe preparation
and denaturation
At the same time
prepare the
a-satellite DNA
probe