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

PRINS and Immunocytochemistry 461
conditions always need to be optimized for the respective instrument used. Because an
accurate temperature at the top surface of the slide is crucial for a successful PRINS
reaction, some cyclers, such as the Hybaid Omnigene, possess incorporated software
to compensate for the temperature difference between the block and the surface of the
slide.
3. Because on methanol-acetone fixed cells we frequently observed a poor preservation of
cell morphology as well as fluorescent staining of the entire nucleus (by PRINS labeling),
probably caused by nuclease activities that survive this mild type of fixation, other fixatives
should be and have been successfully tested that are compatible with antigen detection and
result in a better cell morphology and specific PRINS labeling. For example, a fixation with
cold 70% ethanol (–20°C) for 10 min proved to be a valid alternative on H460 cells.
4. If further amplification of the (immuno)cytochemical signal is needed, a third detection
step may be added after this second incubation step. Alternatively, the avidin biotinylated
enzyme (e.g., alkaline phosphatase) complex system may be applied as well as the tyramide
signal amplification system (in combination with peroxidase conjugates). For details of
possible reagents to use, see Note 13 (7,10,12).
5. It is recommended to monitor the enzyme reaction under the microscope to adjust the
reaction time to ensure the precipitate becoming discretely localized and not so dense that
it shields nucleic acid sequences in the PRINS reaction.
6. To ensure the specificity of the APase-Fast Red staining, a control slide with FITCconjugated
secondary antibodies is recommended for comparison. Staining specificity
can be lost if cells contain endogenous APase activity. This endogenous enzyme activity
can be inhibited by the addition of levamisole (Sigma) to the reaction medium to a final
concentration of 1–5 mM.
7. Do not dehydrate the slides after the APase reaction because the precipitate dissolves in
organic solvents. Optionally, you may air dry the slides after rinsing in distilled water.
8. In the case of labeling with biotin-16-dUTP or fluorescein-12-dUTP a 4× decrease of
the concentration of dTTP in the PRINS reaction mix resulted in significant stronger
labeling of DNA sequences. Under the described standard conditions, digoxigenin-
11-dUTP provides the highest sensitivity. However, all the modified nucleotides are suited
for detection of one or multiple repeated sequences in situ. In this respect, the number of
different fluorochrome- and hapten-labeled nucleotides is still increasing to date, which
can be obtained from a number of companies, such as, e.g., Perkin–Elmer Life Science,
Roche, Amersham, Dako, and Molecular Probes.
9. The concentration of the appropriate oligonucleotide resulting in positive signals need to
be determined by experiment. Generally, 250 ng/slide (50–250 pmol) in 40 µL is used for
primers of 16 to 35 bases complementary to repeated sequences.
10. Seperate denaturation of cellular DNA in 70% formamide/2xSSC, pH 7.0, for 2 min at 70°C
before the PRINS reaction as is usually performed for chromosome preparations, resulted
in no or only weak PRINS labeling of DNA sequences in the interphase nuclei of the cell
preparations. Whether this is caused by inefficient primer annealing or extension is not clear.
The same phenomenon is also observed for PRINS on frozen tissue sections (6,7).
11. The optimum primer annealing temperature is only determined empirically. We usually
try a series from 45 to 70°C in 5°C steps.
12. For detection of multiple DNA targets by sequential PRINS reactions (MULTIPRINS), it
was found essential to prevent the free 3′ ends of the newly synthesized DNA from being
used as primers for subsequent reactions. This can be achieved by incubating the slides
with Klenow DNA polymerase together with ddNTPs. The reaction mix is made up as
follows: Dilute 5 mM of all four ddNTPs 110 with distilled water. Put together in a
centrifuge tube 2.5 µL of each of the ddNTPs (Amersham Pharmacia), 5 µL of 10× Klenow