13 Combined in Situ Nick Translation and mRNA FISH for Simultaneous Detection
173
Preparation of oligonucleotide probes
As probes, DNA oligonucleotides are used. For detecting bcl-2, the 26mer
DNA oligonucleotide probe 5' TCG TAC CCC GTT CTC CCA GCG TGC
GC 3' is used as antisense probe, and the probe 5' GCG CAC GCT GGG
AGA ACG GGG TAC GA 3' as sense probe (Hell et aI., 1995). For detecting
p53, the 20mer DNA oligonucleotide probe 5' TTC TTG CAT TCT GGG
ACA GC 3' was used as antisense probe and the probe 5' GGT TCA AAG
ACC CAA AAC CC 3' as sense probe (PCR primers, MWG Biotech). The
probes are biotinylated at the 3'-end using terminal desoxynucleotidyl
transferase. 100 pmol oligonucleotide is mixed with 2.5 nmol biotin16-dUTP, 1/30 dilution of dATP, 2 ~l cobalt chloride solution and 7.5 U
terminal transferase; subsequent incubation for 15 min at 37°C. The
labeled oligonucleotides are stored at -20°e.
Hybridization
The tissue sections are pre-denatured at 90°C for 15 min and immediately
placed on ice. The hybridization mixture is denatured for 5 min at 95°C
and immediately placed on ice. 50 ~l hybridization mixture is applied to
the slides, covered with coverslips and sealed with rubber cement. After
denaturation for 30 min at 80°C and overnight hybridization at 37°C, the
slides are washed twice in PBS at room temperature.
Detection of apoptosis
Hybridization is followed by in situ nick translation. Fluorescein-12-dUTP
is incorporated in situ into DNA nicks by E. coli polymerase I.
We applied 50 ~l reaction mixture. The slides are covered with coverslips and incubated overnight at 37°e.
Washing steps and detection of mRNA signals
For detection ofbcl-2, the specimens are washed twice in 2xSSC at room
temperature, twice in 2xSSC at 42°C for 10 min and three times in 25%
formamide/2 x SSC at 42°C for 15 min. For detection of p53, the specimens
are washed twice in 2xSSC at room temperature, twice in 2xSSC at 42°C
for 10 min and three times in 33% formamide/2xSSC at 42°C for 15 min.
173
Preparation of oligonucleotide probes
As probes, DNA oligonucleotides are used. For detecting bcl-2, the 26mer
DNA oligonucleotide probe 5' TCG TAC CCC GTT CTC CCA GCG TGC
GC 3' is used as antisense probe, and the probe 5' GCG CAC GCT GGG
AGA ACG GGG TAC GA 3' as sense probe (Hell et aI., 1995). For detecting
p53, the 20mer DNA oligonucleotide probe 5' TTC TTG CAT TCT GGG
ACA GC 3' was used as antisense probe and the probe 5' GGT TCA AAG
ACC CAA AAC CC 3' as sense probe (PCR primers, MWG Biotech). The
probes are biotinylated at the 3'-end using terminal desoxynucleotidyl
transferase. 100 pmol oligonucleotide is mixed with 2.5 nmol biotin16-dUTP, 1/30 dilution of dATP, 2 ~l cobalt chloride solution and 7.5 U
terminal transferase; subsequent incubation for 15 min at 37°C. The
labeled oligonucleotides are stored at -20°e.
Hybridization
The tissue sections are pre-denatured at 90°C for 15 min and immediately
placed on ice. The hybridization mixture is denatured for 5 min at 95°C
and immediately placed on ice. 50 ~l hybridization mixture is applied to
the slides, covered with coverslips and sealed with rubber cement. After
denaturation for 30 min at 80°C and overnight hybridization at 37°C, the
slides are washed twice in PBS at room temperature.
Detection of apoptosis
Hybridization is followed by in situ nick translation. Fluorescein-12-dUTP
is incorporated in situ into DNA nicks by E. coli polymerase I.
We applied 50 ~l reaction mixture. The slides are covered with coverslips and incubated overnight at 37°e.
Washing steps and detection of mRNA signals
For detection ofbcl-2, the specimens are washed twice in 2xSSC at room
temperature, twice in 2xSSC at 42°C for 10 min and three times in 25%
formamide/2 x SSC at 42°C for 15 min. For detection of p53, the specimens
are washed twice in 2xSSC at room temperature, twice in 2xSSC at 42°C
for 10 min and three times in 33% formamide/2xSSC at 42°C for 15 min.