7 FISH on Hair Root Cells
III
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 aI., 1995).
1. Slides with the hair root cells are incubated in 2xSSC for 5 min at RT
(in a 100-ml Coplin jar on a shaker).
2. Remove slides from the Coplin jar, add 100 JlI of RNase solution/slide
and cover with a 24x50 mm coverslip (RNase solution: per slide 100 JlI
2xSSC plus I JlI of RNase stock (= 5 Jlg/Jll).
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment 1).
4. Put slides back into the Coplin jar with 100 ml2 x SSC (RT) and remove
the coverslips with forceps. Leave slides in 2 x SSC solution for 3 min
with gentle agitation.
5. Discard the 2xSSC and replace it with 100 mIl xPBS (RT) for 5 min
(shaker).
6. Replace I xPBS with 100 ml prewarmed pepsin-buffer (37°C) and incubate the slides for 10 min at 37°C, without agitation (see comment 1).
(Pepsin-buffer: Add I ml of 1 M HCI to 99 ml of distilled water and
incubate at 37°C for about 20 min; then add 50 Jll 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/MgClz, incubate at RT for 5 min with
gentle agitation. MgClz will block the enzymatic activity of pepsin
(lxPBS/MgCh = 5% (v/v) 1M MgClz in lxPBS).
8. PostfIX nuclei on the slide surfaces by replacing 1x PBS/MgCI 2 with 100
ml offormalin-buffer (3 ml acid free formaldehyde [37%; Roth 4979.1]
in 100 ml IxPBS) for 10 min (RT, with gentle agitation).
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 2).