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

430 Bull and Paskins
6. Shake the slide free of excess liquid and mount as follows: pipet 40 µL of mountant on
to a clean coverslip and pick up the coverslip with the slide. Place between two layers of
tissue and press to spread the mountant and expel the excess. Seal with rubber solution
(for long term storage) and allow to dry.
7. Slides can be viewed at once or stored for up to a few months in the dark at 4°C without
significant loss of signal. Typical results are shown in Fig. 1.
4. Notes
1. Coated slides are often used in histological procedures where harsh treatments can cause
loss of sample from the slide surface. They can be bought from Fisher or PE Biosystems
or prepared by using poly L-Lysine or aminopropyltriethoxysilane (8). The Amplicover ®
and Ampliclip ® system, together with slides, supplied by PE Biosystems (Foster City,
CA) provides an alternative option for sealing the reaction solution onto the slide surface,
but also requires use of their In Situ PCR 1000 thermal cycler and tool for assembling
the slide chamber. Although relatively expensive, this is the best system for more than
five cycles of PRINS.
2. The relatively high concentration of MgCl 2 is to counteract loss of solution phase Mg
thought to occur during cycling.
3. Alternatively, biotin-labeled dUTP can be used at the same concentration, in which case
a suitable detection reagent is avidin-FITC (Vector Labs). This can be substituted for
anti-DIG FITC, at a 1500 dilution.
4. Most commercially available brands for cycle puncture repair perform adequately. Rema
Tip Top (Munich, Germany) is recommended.
5. Hematological smears work well, as do cytocentrifuge preparations of lymphocytes or
neutrophils or cultured cells. Cells prepared by these methods are flattened and have a
distinct morphological appearance, which is an advantage in interpreting and recording
results because subnuclear spots tend to be in the same focal plane. We have used standard
hematological preparations, such as bone marrow or peripheral blood smears, Giemsastained
slides, and archival slides stored at room temperature, for over a year without
problems. For cultured cells, or leukocyte preparations made from fresh blood (e.g.,
Lymphoprep, Sigma), cytocentrifuge (“cytospin”) preparations are ideal (e.g., Shandon or
Hiraeus cytocentrifuges and equipment). Alternatively, cells can be allowed to settle on
coated slides for 10 to 20 min then allowed to dry after draining off liquid. These methods
are easy to perform, but molecular biologists unfamiliar with cytological preparations
might benefit from a tutorial in a hospital hematology or histology laboratory. Crystallized
solids from support medium or isotonic solutions are not usually problematic because they
are removed in the fixation and microwaving procedure. For extra phenotypic information,
immunocytochemical staining can be done, and signals can be visualized alongside PRINS
signal (7,9).
6. Hygiene precautions (i.e., hand washing) should be taken. The implications of sampling
one’s own blood might merit consideration: An inadvertent diagnosis of aneuploidy may
cause distress! Chromosomes 3, 7, and 17 should be safe in this regard.
7. Ethanol fixation works by precipitating proteins irreversibly from solution. Other precipitating
fixatives we have successfully used include methanol and acetone.
8. Reactions can be performed under coverslips or using the PE Applied Biosystems system
(Amplicovers and Ampliclips). If using coverslips, some slides may start to dry out after
around 10 cycles, but this will depend on the size of your coverslip and the type of rubber
solution used to seal it to the slide. We find that 5 to 7 cycles is a good compromise. Using
the PE system, we have cycled up to 70 times.