11 Formalin-Fixed and Paraffin-Embedded Tissue Sections
159
tend to destroy cell structure and tissue morphology. However, mild pretreatment saving tissue structures, may not be sufficient for probe penetration and successful FISH results. Especially locus specific probes with a
single copy target may show dim or a reduced number of signals.
Generally: If the experiment did not work - before changing any of the
parameters, try it again!
Problem
Presumed Cause
Solution
Porous
paraffin
block
- Prolonged storage
- Embed anew
Use positively charged (e.g. Super Frost
Menzel) or coated slides (e.g. poly-I-lysine, silane) and bake the slides at 56°C
overnight - Check temperature of
NaSCN buffer (should not exceed 80°C)
- Decrease the incubation time of NaSCN
- Increase the incubation time of buffered
formalin (some specimens do not stick
to slides at all!)
- Insufficient slide coating -
- Tissue gets lost during
the NaSCN procedure
- The tissue was not flxed
thoroughly
Tissue loss,
complete
or partial
Poor tissue
morphology
High tissue
autofluorescence
- The tissue was digested - Reduce the incubation time in protease
too extensively
solution
- The tissue was exten- Check temperature of the protease sosively fixed (specimen
lution
probably exceeded fixa- - Eliminate or reduce the fixation step
tion times of 24-48 h)
with formalin
- The tissue was insuffi- - Choose new specimen
ciently fixed or autolytic - Choose new specimen
- Choose new specimen, problem cannot
be eliminated
Fig.3. Dual color FISH with differently labeled DNA probes for the HER-2/neu gene (red),
localized on the long arm of chromosome 17, and for centromeric alpha satellite DNA
(green) of chromosome 17, performed on sections of formalin-fixed archival breast cancer
tissue. a Multiple distinct signals of HER-2/neu as well as of centromeric DNA of chromosome 17 point to polysomies ofchromosome 17. b On the right, red signals appear as clusters
demonstrating high amplifications of HER-2/neu in tumor cells. Distinct green signals indicate multiple copies of chromosome 17. The HER-2/neu amplifications are estimated at
10-40 signals per nucleus. On the left, normal connective tissue with vascular structures is
visible showing normal signal patterns.
159
tend to destroy cell structure and tissue morphology. However, mild pretreatment saving tissue structures, may not be sufficient for probe penetration and successful FISH results. Especially locus specific probes with a
single copy target may show dim or a reduced number of signals.
Generally: If the experiment did not work - before changing any of the
parameters, try it again!
Problem
Presumed Cause
Solution
Porous
paraffin
block
- Prolonged storage
- Embed anew
Use positively charged (e.g. Super Frost
Menzel) or coated slides (e.g. poly-I-lysine, silane) and bake the slides at 56°C
overnight - Check temperature of
NaSCN buffer (should not exceed 80°C)
- Decrease the incubation time of NaSCN
- Increase the incubation time of buffered
formalin (some specimens do not stick
to slides at all!)
- Insufficient slide coating -
- Tissue gets lost during
the NaSCN procedure
- The tissue was not flxed
thoroughly
Tissue loss,
complete
or partial
Poor tissue
morphology
High tissue
autofluorescence
- The tissue was digested - Reduce the incubation time in protease
too extensively
solution
- The tissue was exten- Check temperature of the protease sosively fixed (specimen
lution
probably exceeded fixa- - Eliminate or reduce the fixation step
tion times of 24-48 h)
with formalin
- The tissue was insuffi- - Choose new specimen
ciently fixed or autolytic - Choose new specimen
- Choose new specimen, problem cannot
be eliminated
Fig.3. Dual color FISH with differently labeled DNA probes for the HER-2/neu gene (red),
localized on the long arm of chromosome 17, and for centromeric alpha satellite DNA
(green) of chromosome 17, performed on sections of formalin-fixed archival breast cancer
tissue. a Multiple distinct signals of HER-2/neu as well as of centromeric DNA of chromosome 17 point to polysomies ofchromosome 17. b On the right, red signals appear as clusters
demonstrating high amplifications of HER-2/neu in tumor cells. Distinct green signals indicate multiple copies of chromosome 17. The HER-2/neu amplifications are estimated at
10-40 signals per nucleus. On the left, normal connective tissue with vascular structures is
visible showing normal signal patterns.