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NORBERT HECKMANN
Materials
Hybridization mixture
- 50% Formamide
- 2xSSC
- 10% dextran sulphate
- 1 J.lI E. coli tRNA
- 1 J.lI sheared salmon sperm DNA
- A dilution of 1/50 of labeled oligonucleotide probes
- add 50 J.lI with double distilled water
Polymerase-enzyme-mix
- 0.5 nmol of each dNTP
- 0.25 nmol fluorescein-12-dUTP
- 5 U DNA polymerase I (endonuclease-free)
- add 50 J.lI with double distilled water
,,; Procedure
Preparation of paraffin-embedded sections
We use sections from tissue routinely fixed in 4% buffered formaldehyde
(pH 7.4) and embedded in paraffin. For the pretreatment, all solutions are
prepared with double distilled water containing 0.1% diethyl pyrocarbonate (DEPC) to avoid RNase activity. The 5 J.lm thick sections are dewaxed
with xylene and rehydrated in a graded ethanol series down to 50%, and
the slides are dried at 50°C. After postfIXing for 10 min (4% paraformaldehyde) and washing (PBS-phosphate buffered saline, water) they are incubated in proteinase K solution (20 J.lglml proteinase K in 50 mM trisHCI, pH 7.6; 5 mM EDTA) for 15-20 min at 55°C. Washing for 5 min (PBS,
water) is followed by incubation in 2xSSC (standard saline citrate) at 70°C
for 20 min. The sections are dehydrated in an ethanol series of increasing
concentration and air-dried.
NORBERT HECKMANN
Materials
Hybridization mixture
- 50% Formamide
- 2xSSC
- 10% dextran sulphate
- 1 J.lI E. coli tRNA
- 1 J.lI sheared salmon sperm DNA
- A dilution of 1/50 of labeled oligonucleotide probes
- add 50 J.lI with double distilled water
Polymerase-enzyme-mix
- 0.5 nmol of each dNTP
- 0.25 nmol fluorescein-12-dUTP
- 5 U DNA polymerase I (endonuclease-free)
- add 50 J.lI with double distilled water
,,; Procedure
Preparation of paraffin-embedded sections
We use sections from tissue routinely fixed in 4% buffered formaldehyde
(pH 7.4) and embedded in paraffin. For the pretreatment, all solutions are
prepared with double distilled water containing 0.1% diethyl pyrocarbonate (DEPC) to avoid RNase activity. The 5 J.lm thick sections are dewaxed
with xylene and rehydrated in a graded ethanol series down to 50%, and
the slides are dried at 50°C. After postfIXing for 10 min (4% paraformaldehyde) and washing (PBS-phosphate buffered saline, water) they are incubated in proteinase K solution (20 J.lglml proteinase K in 50 mM trisHCI, pH 7.6; 5 mM EDTA) for 15-20 min at 55°C. Washing for 5 min (PBS,
water) is followed by incubation in 2xSSC (standard saline citrate) at 70°C
for 20 min. The sections are dehydrated in an ethanol series of increasing
concentration and air-dried.