John M. S. Bartlett.pdf - Bio-Nica.info

Direct and Indirect In Situ PCR 441
of 200 to 800 bp because with larger amplicons the amplification efficiency will be reduced
dramatically and will often result in false negative samples.
5. Because dextran sulfate may produce background staining, especially if higher concentrations
are used, reduce the amount of dextran sulfate to diminish the background staining.
For ISH in contrast to filter hybridization, usually 10% dextran sulfate is the upper limit.
6. The reverse transcription can also be performed with other transcriptases, such as
Superscript (GibcoBRL, Karlsruhe, Germany). In this case, the protocol has to be adjusted
accordingly.
7. For SuperFrostPlus slides, this is only a precaution as when stored (dust free) and
handled under appropriate conditions, we have encountered neither DNAse nor RNAse
contamination.
8. Coverslips may be siliconized prior to the incubation at 220°C if adherence of tissue
sample to the coverslips is observed during the PCR procedure.
9. To obtain reproducible results, it is essential that fixation is always performed at the same
temperature and for the same time. Otherwise, the permeabilization conditions have to
be adapted and optimized for each PCR. A difference of 5°C during fixation may give
false negative results because of changes in cell permeability. Unfortunately, tissue in
molecular pathology is usually not fixed under standardized conditions. Therefore, several
permeabilization conditions have to be tested for each sample.
To improve fixation and therefore DNA and RNA preservation, tissues should be cut
to the minimum size prior to fixation.
For mRNA detection paraformaldehyde will yield a better preservation of RNA than
formalin. Perform fixation at 4°C where possible. The extraction of RNA from formalinor
paraformaldehyde-fixed tissues for doing a solution-phase PCR to verify the in situ
results especially when establishing a new protocol is usually very ineffective. For these
applications, we recommend the use of the Hope fixative (patent pending, Dr. Olert, Institut für
Kinderpathologie, Universität Mainz, Germany available at DCS, Hamburg, Germany) which
additionally yields a much better preservation of RNA compared to other fixatives.
10. Thinner sections show better adhesion to slides during IS-PCR, although this reduces the
number of target sequences, which may result in some negative cells.
11. Use fresh reagents for each incubation. Because xylenes saturate rapidly with paraffin use
200 mL of fresh xylene for each batch of 15 to 20 slides.
12. Although fixed under the same conditions, different tissues may require adapted permeabilization
parameters. When optimizing permeabilization parameters with respect to
morphology, it is usually better to prolong Proteinase K incubation than to increase the
concentration of Proteinase K (15). If diffusion artifacts are a major problem the reduction
of the concentration of Proteinase K with simultaneous prolongation of incubation turned
out to be advantageous (15). If you encounter background staining this may be reduced
by the use of target retrieval instead of Proteinase K (15). Proteinase K (250 µg/ mL) is
optimal for portio and condylomata biopsies fixed for 24 h in buffered formalin whereas
for leukocytes 10 to 30 µg/mL Proteinase K and for bronchial epithelial cells 30 to
50 µg/mL Proteinase K performed well. Leukocytes and bronchial epithelial cells were
fixed with 4% buffered paraformaldehyde for 30 min.
13. The concentration of endogenous peroxidase varies significantly between tissues. Leukocytes
exhibiting high endogenous peroxidase levels usually require longer incubation
(45 min) than other tissues. If you encounter signals in your negative controls try to
diminish those signals either by prolongation of the quenching reaction or by increasing
the concentration of H 2 O 2 up to 3%.
14. Evaporation control is of the utmost importance for reliable and reproducible results as
unspecific signals because of the evaporation of the reaction mixture may result from insuf-