162
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Paraffinembedded
tissue sections
glutaraldehyde: incubate the coated slides in 2.5% glutaraldehyde dissolved
in phosphate-buffered saline (PBS) for 1 h at room temperature then wash
in PBS and water. (As an alternative one can also use alkylamine coated
slides).
1. Paraffin sections (about 4-6 J..lm thick) in water (40°C) are mounted on
glutaraldehyde-activated-poly-L-lysine coated slides.
2. Air dry and hake slides at 56°C overnight.
3. Deparaffinate 3 times in 100% xylene for 10 min and twice in absolute
methanol for 5 min. each.
4. Immerse slides in 1% H20 2 dissolved in absolute methanol for 30 min.
5. Wash twice in 100% methanol for 5 min then air dry.
6. Immerseslides in 1M sodium thiocyanate at 80°C for 10 min (this step is
optional, see above).
7. Wash twice in water for 5 min.
8. Incubate slides 5- 60 min at 37°C in digestion solution. If sections were
incubated with sodium thiocyanate (step 6) reduce incubation time to
5-15 min.
9. Wash 5 times in water, then 5 times in PBS briefly.
10. Dehydrate the sections in 70%, 90% and 100% ethanol for 3 min, then
air dry.
11. Denature slides in denaturation solution at 80°C for 4-10 min in a Coplin jar (see note below).
12. Immediately dehydrate in ice-cold 70%, 90% and 100% ethanol (5 min
each) and add hybridization solution containing denaturated probe.
13. Hybridize overnight at 37°C in a humidified chamber, then detect signals as described in Chap. 12.
Note: Denaturation of the probe as weil as the target DNA can be performed
in a single step. After adding the probe solved in 10-15 J..ll ofhybridization
solution to the tumor cells the slide is heated on a warm plate for about 4-10
min at 80°C.
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Paraffinembedded
tissue sections
glutaraldehyde: incubate the coated slides in 2.5% glutaraldehyde dissolved
in phosphate-buffered saline (PBS) for 1 h at room temperature then wash
in PBS and water. (As an alternative one can also use alkylamine coated
slides).
1. Paraffin sections (about 4-6 J..lm thick) in water (40°C) are mounted on
glutaraldehyde-activated-poly-L-lysine coated slides.
2. Air dry and hake slides at 56°C overnight.
3. Deparaffinate 3 times in 100% xylene for 10 min and twice in absolute
methanol for 5 min. each.
4. Immerse slides in 1% H20 2 dissolved in absolute methanol for 30 min.
5. Wash twice in 100% methanol for 5 min then air dry.
6. Immerseslides in 1M sodium thiocyanate at 80°C for 10 min (this step is
optional, see above).
7. Wash twice in water for 5 min.
8. Incubate slides 5- 60 min at 37°C in digestion solution. If sections were
incubated with sodium thiocyanate (step 6) reduce incubation time to
5-15 min.
9. Wash 5 times in water, then 5 times in PBS briefly.
10. Dehydrate the sections in 70%, 90% and 100% ethanol for 3 min, then
air dry.
11. Denature slides in denaturation solution at 80°C for 4-10 min in a Coplin jar (see note below).
12. Immediately dehydrate in ice-cold 70%, 90% and 100% ethanol (5 min
each) and add hybridization solution containing denaturated probe.
13. Hybridize overnight at 37°C in a humidified chamber, then detect signals as described in Chap. 12.
Note: Denaturation of the probe as weil as the target DNA can be performed
in a single step. After adding the probe solved in 10-15 J..ll ofhybridization
solution to the tumor cells the slide is heated on a warm plate for about 4-10
min at 80°C.