14 Nucleus Extraction from Cryoflxed Tissue
177
fIxation is performed in unbuffered formalin andlor incubation is too
long, the tissue becomes unsuitable for any kind of FISH studies, because
DNA is degraded and washed out of the cells (Long et aI., 1993). When
using sectioned material, some additional problems arise. The most important ones arise during evaluation of the slides: (a) there is the problem
that overlapping nuclei, due to the presence of several cell layers, one on
top of the other, cannot be evaluated; and (b) the problem that cut nuclei
may lead to artifIcial signal loss in interphase cytogenetic studies (Neubauer et aI., 1994, Dhingra et aI., 1992).
These problems can be solved using a technique called nuclear extraction, fIrst described in 1983 (Hedley et aI., 1983). However, there is only
one protocol concerning nuclear extraction from cryofIxed tissues in the
literature (Liehr et aI., 1996, 1997).
Outline
1. CryofIxed tissue is cut into small pieces whilst still frozen, formalinfIxed, washed and treated with proteinase K to digest the cytoplasm
and free nuclei. During this treatment, damaged and cut nuclei are
eliminated. Interphase nuclei are separated from debris by passing
through a nylon mesh. Nuclei are washed in PBS and placed on uncoated, clean slides and fIxed. Small tissue pieces (5x2x0.5 mm) are
suffIcient to produce two to fIve slides with extracted nuclei suitable for
interphase FISH analyses.
2. For a FISH approach, these slides are treated with RNase and pepsin
prior to denaturation.
3. Denaturation of the slides is performed for about 12 min at 75°C. Biotinylated and digoxigenated probes are denatured for 5 min and hybridized on the slides for 3 nights at 37°C in a humid chamber. After a
postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to a green signal and of
the digoxigenated probe with anti-digoxigenin-rhodamine leading
to red signals. After counterstaining of the nuclei with DAPI and addition of antifade solution, slides can be evaluated under a fluorescence microscope.
177
fIxation is performed in unbuffered formalin andlor incubation is too
long, the tissue becomes unsuitable for any kind of FISH studies, because
DNA is degraded and washed out of the cells (Long et aI., 1993). When
using sectioned material, some additional problems arise. The most important ones arise during evaluation of the slides: (a) there is the problem
that overlapping nuclei, due to the presence of several cell layers, one on
top of the other, cannot be evaluated; and (b) the problem that cut nuclei
may lead to artifIcial signal loss in interphase cytogenetic studies (Neubauer et aI., 1994, Dhingra et aI., 1992).
These problems can be solved using a technique called nuclear extraction, fIrst described in 1983 (Hedley et aI., 1983). However, there is only
one protocol concerning nuclear extraction from cryofIxed tissues in the
literature (Liehr et aI., 1996, 1997).
Outline
1. CryofIxed tissue is cut into small pieces whilst still frozen, formalinfIxed, washed and treated with proteinase K to digest the cytoplasm
and free nuclei. During this treatment, damaged and cut nuclei are
eliminated. Interphase nuclei are separated from debris by passing
through a nylon mesh. Nuclei are washed in PBS and placed on uncoated, clean slides and fIxed. Small tissue pieces (5x2x0.5 mm) are
suffIcient to produce two to fIve slides with extracted nuclei suitable for
interphase FISH analyses.
2. For a FISH approach, these slides are treated with RNase and pepsin
prior to denaturation.
3. Denaturation of the slides is performed for about 12 min at 75°C. Biotinylated and digoxigenated probes are denatured for 5 min and hybridized on the slides for 3 nights at 37°C in a humid chamber. After a
postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to a green signal and of
the digoxigenated probe with anti-digoxigenin-rhodamine leading
to red signals. After counterstaining of the nuclei with DAPI and addition of antifade solution, slides can be evaluated under a fluorescence microscope.