14 Nucleus Extraction from Cryofixed Tissue
179
7. Repeat step 5.
8. Repeat steps 6 and 7.
9. Remove the fluid using a 200 JlI Eppendorf pipette, add - depending on
the amount of tissue - 0.2-1 ml of PK-solution and vortex the microtube [PK-solution: 5 mg proteinase K (Boehringer 745723),50 JlI 1 M
tris-HCI (pH 7.5), 20 JlI 0.5 M EDTA (pH 7.0), 2 Jll5 M NaCI, make up to
1 ml with fIltered double distilled water; make fresh as required] (see
comment 3 as well).
10. Incubate the microtube at 37°C for 30 min. During this time, vortex the
microtube every 5 min to promote tissue disaggregation.
11. Transfer the fluid onto a 55 Jlm nylon mesh (Nytal 55 (SEFAR-AG,
ThaI, Switzerland; see comment 4). 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.
12. Pellet the extracted nuclei by centrifugation (850 g, 8 min); remove the
supernatant with the exception of about 300 Jll.
13. Resuspend the pellet in 4 ml I xPBS and repeat step 12.
14. Resuspend the remaining 300 JlI of 1x PBS.
15. Distribute the suspension on 2-6 clean dry slides by pipetting the fluid
onto the slide surfaces. Place one drop (30-90 JlI ) of about 1 cm in
diameter/slide (see comment 5).
16. Allow drops to dry out on a 40°C warming plate and afterwards at RT
overnight.
17. Fix slides in 100 ml formalin buffer in a Coplin jar for 10 min (RT).
18. Replace formalin-buffer in the Coplin jar with 1x PBS. After 5 min of
incubation at RT, 1x PBS is replaced by distilled water.
19. Remove the water after 1 min, perform an ethanol series (70%, 90%,
100%, 3 min each) to dehydrate slides and air dry. An evaluation of the
success of the nuclear extraction can be performed by phase contrast
light microscopy (see comment 6).
179
7. Repeat step 5.
8. Repeat steps 6 and 7.
9. Remove the fluid using a 200 JlI Eppendorf pipette, add - depending on
the amount of tissue - 0.2-1 ml of PK-solution and vortex the microtube [PK-solution: 5 mg proteinase K (Boehringer 745723),50 JlI 1 M
tris-HCI (pH 7.5), 20 JlI 0.5 M EDTA (pH 7.0), 2 Jll5 M NaCI, make up to
1 ml with fIltered double distilled water; make fresh as required] (see
comment 3 as well).
10. Incubate the microtube at 37°C for 30 min. During this time, vortex the
microtube every 5 min to promote tissue disaggregation.
11. Transfer the fluid onto a 55 Jlm nylon mesh (Nytal 55 (SEFAR-AG,
ThaI, Switzerland; see comment 4). 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.
12. Pellet the extracted nuclei by centrifugation (850 g, 8 min); remove the
supernatant with the exception of about 300 Jll.
13. Resuspend the pellet in 4 ml I xPBS and repeat step 12.
14. Resuspend the remaining 300 JlI of 1x PBS.
15. Distribute the suspension on 2-6 clean dry slides by pipetting the fluid
onto the slide surfaces. Place one drop (30-90 JlI ) of about 1 cm in
diameter/slide (see comment 5).
16. Allow drops to dry out on a 40°C warming plate and afterwards at RT
overnight.
17. Fix slides in 100 ml formalin buffer in a Coplin jar for 10 min (RT).
18. Replace formalin-buffer in the Coplin jar with 1x PBS. After 5 min of
incubation at RT, 1x PBS is replaced by distilled water.
19. Remove the water after 1 min, perform an ethanol series (70%, 90%,
100%, 3 min each) to dehydrate slides and air dry. An evaluation of the
success of the nuclear extraction can be performed by phase contrast
light microscopy (see comment 6).