4 FISH on Uncultured Blood or Bone Marrow
85
7. Repeat step 2.
8. Resuspend the pellet in 5 ml of fIxative (4°C) and repeat step 2.
9. Repeat step 8 twice.
10. According to the density of the suspension, the pellet is fInally resuspended in 0.3-1 ml of fIxative (suck off as much of the suspension as
necessary after step 9).
11. Bring 1 to 2 drops of the suspension, each, on a clean and humid slide
with a glass pipette and let the slide dry at RT.
12. After an incubation over night at RT, the slides can be subjected to the
pretreatment (see below), stored dust free at RT for several weeks or
frozen at -20°C for several months.
Slide pretreatment
1. Slides with the interphase 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 JlI of RNase solution/slide
and cover with a 24x50 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 I).
4. Put slides back into the Coplin jar with 100 ml 2x 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 I).
(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 x PBS/MgC!z, incubate at RT for 5 min with
gentle agitation. MgClz will block the enzymatic activity of pepsin
(lxPBS/MgC!z =5% (v/v) 1M MgC!z in 1xPBS).