164
THOMAS LIEHR
postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to green signals 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.
. Procedure
Nuclear extraction
Nuclear extraction
from unmounted
formalin-fixed/paraffin-embedded
tissues
1. According to the diameter of the piece of tissue studied, two to twenty
20 /Jm sections of paraffin-embedded tissue are produced on a microtome and collected in a glass tube.
2. Dewax the tissue pieces by adding 10 ml ofxylene (100%), for 10 min at
room temperature (=RT).
3. Tissue is sedimented by centrifugation (1000 rpm, 3 min) and the
supernatant is discarded (1-2 ml of supernatant are left in the tube
to avoid loss of small tissue pieces).
4. Repeat step 2 and 3.
5. Remove the xylene rest by adding 10 ml of ethanol (100%) and incubate for 10 min at RT.
6. Repeat step 3.
7. Repeat step 5 and 6.
8. Rehydrate the tissue by adding 10 ml of 90% ethanol (5 min at RT),
10 ml ethanol 70% (5 min at RT), 10 ml ethanol 50% (5 min at RT) and
10 ml 0.9% NaCl (5 min at RT). Remove the corresponding supernatants by repeating step 3.
9. Wash the rehydrated tissue in 10 ml 0.9% NaCl (2 min at RT) and repeat step 3.
10. Put the tissue, together with appr. 1 ml of the NaCl solution from the
glass tube into a 1.5-ml microtube. This can easily be done using a I-ml
Eppendorf pipette with a cut blue tip, thus enhancing the diameter of
the tip.
THOMAS LIEHR
postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to green signals 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.
. Procedure
Nuclear extraction
Nuclear extraction
from unmounted
formalin-fixed/paraffin-embedded
tissues
1. According to the diameter of the piece of tissue studied, two to twenty
20 /Jm sections of paraffin-embedded tissue are produced on a microtome and collected in a glass tube.
2. Dewax the tissue pieces by adding 10 ml ofxylene (100%), for 10 min at
room temperature (=RT).
3. Tissue is sedimented by centrifugation (1000 rpm, 3 min) and the
supernatant is discarded (1-2 ml of supernatant are left in the tube
to avoid loss of small tissue pieces).
4. Repeat step 2 and 3.
5. Remove the xylene rest by adding 10 ml of ethanol (100%) and incubate for 10 min at RT.
6. Repeat step 3.
7. Repeat step 5 and 6.
8. Rehydrate the tissue by adding 10 ml of 90% ethanol (5 min at RT),
10 ml ethanol 70% (5 min at RT), 10 ml ethanol 50% (5 min at RT) and
10 ml 0.9% NaCl (5 min at RT). Remove the corresponding supernatants by repeating step 3.
9. Wash the rehydrated tissue in 10 ml 0.9% NaCl (2 min at RT) and repeat step 3.
10. Put the tissue, together with appr. 1 ml of the NaCl solution from the
glass tube into a 1.5-ml microtube. This can easily be done using a I-ml
Eppendorf pipette with a cut blue tip, thus enhancing the diameter of
the tip.