30 Microdissection of Chromosomes and Reverse FISH
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mix that was prepared accordingly can be added instead. The probe is
denatured at 75
D C for 5 min, chilled down rapidly (e.g. ice water) and incubated at 37
D C for 30 min.
Slide preparation
Conventional chromosome slides can be used for the hybridization. However, slides which have been spread on the same day the hybridization is to
be carried out, are not suitable. Chromosome morphology is better retained if slides are aged at least overnight. Pretreatment of chromosome
slides prior to the hybridization is optional. However, RNase digestion
might reduce the background staining and digestion with a proteolytic
enzyme (Pepsin or Proteinase K) improves the probe penetration. Slides
should not be older than 2 weeks.
Chromosome denaturation
Denaturation solution
- 0.7 ml Formamide
- 0.1 ml20xSSC
- 0.2 ml H 2 0
- pH 7.0
1. Prewarm a heating plate to 70
D
e.
2. Place slides on the heating plate.
3. Apply 100 III denaturation solution to each slide.
4. Overlay with a 24x60 mm coverslip.
5. Incubate for 3 min at 70
D
e.
6. Flip off the coverslip and.
7. Immediately transfer to a Coplin jar with 70% ethanol for 1 min.
8. Subsequently, transfer to a Coplin jar with 90% and 100% ethanol, incubate for 1 min each at RT.
9. Air dry.