166
THOMAS LIEHR
4. Resuspend the remaining 300 III of 1x PBS.
5. Distribute the suspension on 2-6 clean dry slides by pipetting the fluid
onto the slide surfaces. Place one drop (30-90 III ) of about 1 cm in
diameter/slide (see comment 2).
6. Allow drops to dry out on a 40°C warming plate and afterwards incubate at RT overnight.
7. Fix slides in 100 ml formalin buffer in a Coplin jar for 10 min (RT).
8. Replace formalin-buffer in the Coplin jar with 1x PBS. After 5 min of
incubation at RT, 1xPBS is replaced by distilled water.
9. 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 3).
Slide pretreatment
As in a conventional FISH approach, a pretreatment of the slides with
RNase and pepsin followed by a postflXation with formalin-buffer is required to reduce the background (Liehr et al. 1995b).
1. Slides with the extracted nuclei are incubated in 2x SSC for 5 min at RT
(in a 100-ml Coplin jar on a shaker).
2. Remove slides from the Coplin jar, add 100 III of RNase solution/slide
and cover with a 24xSO mm coverslip (RNase solution: per slide 100 III
2xSSC plus 1 III of RNase stock (= 5 Ilg/Ill».
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment 4).
4. Put slides back into the Coplin jar with 100 ml2 x SSC (RT) and remove
the coverslips with forceps. Leave slides in 2x SSC solution for 3 min
with gentle agitation.
5. Discard 2xSSC and replace it with 100 mIl xPBS (RT) for 5 min (shaker).
6. Replace 1xPBS with 100 ml prewarmed pepsin-buffer (37°C) and incubate the slides for 10 min at 37°C, without agitation (see comment 4).
(Pepsin-buffer: Add 1 ml of 1 M HCI to 99 ml of distilled water and
THOMAS LIEHR
4. Resuspend the remaining 300 III of 1x PBS.
5. Distribute the suspension on 2-6 clean dry slides by pipetting the fluid
onto the slide surfaces. Place one drop (30-90 III ) of about 1 cm in
diameter/slide (see comment 2).
6. Allow drops to dry out on a 40°C warming plate and afterwards incubate at RT overnight.
7. Fix slides in 100 ml formalin buffer in a Coplin jar for 10 min (RT).
8. Replace formalin-buffer in the Coplin jar with 1x PBS. After 5 min of
incubation at RT, 1xPBS is replaced by distilled water.
9. 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 3).
Slide pretreatment
As in a conventional FISH approach, a pretreatment of the slides with
RNase and pepsin followed by a postflXation with formalin-buffer is required to reduce the background (Liehr et al. 1995b).
1. Slides with the extracted nuclei are incubated in 2x SSC for 5 min at RT
(in a 100-ml Coplin jar on a shaker).
2. Remove slides from the Coplin jar, add 100 III of RNase solution/slide
and cover with a 24xSO mm coverslip (RNase solution: per slide 100 III
2xSSC plus 1 III of RNase stock (= 5 Ilg/Ill».
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment 4).
4. Put slides back into the Coplin jar with 100 ml2 x SSC (RT) and remove
the coverslips with forceps. Leave slides in 2x SSC solution for 3 min
with gentle agitation.
5. Discard 2xSSC and replace it with 100 mIl xPBS (RT) for 5 min (shaker).
6. Replace 1xPBS with 100 ml prewarmed pepsin-buffer (37°C) and incubate the slides for 10 min at 37°C, without agitation (see comment 4).
(Pepsin-buffer: Add 1 ml of 1 M HCI to 99 ml of distilled water and