180
BERND RAUTENSTRAUSS and THOMAS LIEHR
Slide pretreatment
As in a conventional FISH approach, a pretreatment of the slides with
RNase and pepsin followed by a postfIxation with formalin-buffer is required to reduce background (Liehr et al., 1995).
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 I.d of RNase solution/slide
and cover with a 24 x 50 mm coverslip [RNase solution: per slide 100 JlI
2xSSC plus 1 JlI of RNase stock (= 5 Jlg/JlI)].
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment 7).
4. Put slides back into the Coplin jar with 100 ml2xSSC (RT) and remove
the coverslips with forceps. Leave slides in 2 x 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 7).
[Pepsin-buffer: Add 1 ml of 1 M HCI to 99 ml of distilled water and
incubate at 37°C for about 20 min; then add 50 JlI of the pepsin stock
solution 10% (w/v) (Serva 31855) and leave the Coplin jar at 37°C;
make fresh as required].
7. Replace fluid with 100 mIl xPBS/MgCh, incubate at RT for 5 min with
gentle agitation. MgCh will block the enzymatic activity of pepsin.
8. Postfix nuclei on the slide surfaces by replacing 1x PBS/MgCl z with
100 ml of formalin-buffer for 10 min (RT, with gentle agitation).
(lxPBS/ MgCh=5% (v/v) 1M MgCh in lxPBS).
9. Formalin-buffer is replaced by 100 mIl x PBS for 2 min (RT, with gentle agitation).
10. Finally, slides are dehydrated by an ethanol series (70%, 90%, 100%,
3 min each) and air dried (see comment 8).