12 Nucleus Extraction from Formalin Fixed/paraffin Embedded Tissue
165
11. Remove the NaCl solution from the microtube using a 200-1l1 Eppendorf pipette, add - depending on the amount of tissue - 0.2-1 ml of PKsolution and vortex the microtube (PK-solution: 5 mg proteinase K
(Boehringer 745723), 50 III 1 M Tris-HCl (pH 7.5), 20 III 0.5 M
EDTA (pH 7.0), 2 III 5 M NaCI, make up to 1 ml with filtered double
distilled water; make fresh as required).
12. Incubate the microtube at 37°C for 30 min. During this time, vortex the
microtube every 5 min to promote tissue disaggregation.
13. Go on with the protocol "Purification of the released nuclei".
1. A 5 Ilm section of paraffin-embedded tissue is mounted on a glass
slide. Uncoated or coated slides (e.g. with aminotriethoxysilane or
polylysine) can be used.
2. The mounted section is deparaffinized in 100 ml xylene in a Coplin jar
(two times 5 min) and rehydrated in an ethanol series (100%, 90%,
70%, 50%; 3 min each) and 0.9% NaCI solution (two times 2 min).
3. If necessary, undesirable parts of the tissue can be removed at this
point by scratching it from the slide with a scalpel. This may be helpful
when analyzing tumors infIltrating normal tissue.
4. After that, the tissue is covered with proteinase K solution (PK-solution: 5 mg proteinase K (Boehringer 745723), 50 III 1M Tris-HCI (pH
7.5),20 III 0.5 M EDTA (pH 7.0),21115 M NaCI, make up to 1 ml with
fIltered double distilled water; make fresh as required) and incubated
at 37°C for approx. 1 h in a moist chamber. The use of a coverslip
should be avoided.
5. Thereafter, the proteinase K solution together with the digested tissue
and released nuclei is collected with a pipette and passed through a
55 Ilm nylon mesh (go on with the following protocol).
1. Transfer the fluid onto a 55 Ilm nylon mesh (Nytal 55 (SEFAR-AG,
ThaI, Switzerland; see comment 1). Fluid and nuclei will pass through
the mesh by force of gravity and are collected in a 15-ml plastic tube.
Nuclei remaining in the mesh are washed out by 4 ml 1x PBS, passed
through the mesh and collected in the 15 ml plastic tube.
2. Pellet the extracted nuclei by centrifugation (850 g, 8 min); remove the
supernatant leaving about 300 Ill.
3. Resuspend the pellet in 4 ml 1x PBS and repeat step 2.
Nuclear extraction
from mounted formalin fixed/paraffin
embedded tissues
Purification of the
released nuclei
165
11. Remove the NaCl solution from the microtube using a 200-1l1 Eppendorf pipette, add - depending on the amount of tissue - 0.2-1 ml of PKsolution and vortex the microtube (PK-solution: 5 mg proteinase K
(Boehringer 745723), 50 III 1 M Tris-HCl (pH 7.5), 20 III 0.5 M
EDTA (pH 7.0), 2 III 5 M NaCI, make up to 1 ml with filtered double
distilled water; make fresh as required).
12. Incubate the microtube at 37°C for 30 min. During this time, vortex the
microtube every 5 min to promote tissue disaggregation.
13. Go on with the protocol "Purification of the released nuclei".
1. A 5 Ilm section of paraffin-embedded tissue is mounted on a glass
slide. Uncoated or coated slides (e.g. with aminotriethoxysilane or
polylysine) can be used.
2. The mounted section is deparaffinized in 100 ml xylene in a Coplin jar
(two times 5 min) and rehydrated in an ethanol series (100%, 90%,
70%, 50%; 3 min each) and 0.9% NaCI solution (two times 2 min).
3. If necessary, undesirable parts of the tissue can be removed at this
point by scratching it from the slide with a scalpel. This may be helpful
when analyzing tumors infIltrating normal tissue.
4. After that, the tissue is covered with proteinase K solution (PK-solution: 5 mg proteinase K (Boehringer 745723), 50 III 1M Tris-HCI (pH
7.5),20 III 0.5 M EDTA (pH 7.0),21115 M NaCI, make up to 1 ml with
fIltered double distilled water; make fresh as required) and incubated
at 37°C for approx. 1 h in a moist chamber. The use of a coverslip
should be avoided.
5. Thereafter, the proteinase K solution together with the digested tissue
and released nuclei is collected with a pipette and passed through a
55 Ilm nylon mesh (go on with the following protocol).
1. Transfer the fluid onto a 55 Ilm nylon mesh (Nytal 55 (SEFAR-AG,
ThaI, Switzerland; see comment 1). Fluid and nuclei will pass through
the mesh by force of gravity and are collected in a 15-ml plastic tube.
Nuclei remaining in the mesh are washed out by 4 ml 1x PBS, passed
through the mesh and collected in the 15 ml plastic tube.
2. Pellet the extracted nuclei by centrifugation (850 g, 8 min); remove the
supernatant leaving about 300 Ill.
3. Resuspend the pellet in 4 ml 1x PBS and repeat step 2.
Nuclear extraction
from mounted formalin fixed/paraffin
embedded tissues
Purification of the
released nuclei