92
THOMAS LIEHR
Slide pretreatment
During the whole slide pretreatment procedure, the slides lose the red
erythrocytes and they become more and more transparent. At the end
(step 10), the nuclei of the blasts and lymphocytes are the only remaining
cellular components on the slide surface. If the cellular structure is to be
maintained and tumor cells are to be distinguished from nontumor cells,
this slide pretreatment step must be replaced by a lO-min incubation of
the slides in 2x SSC at room temperature, prior to the FISH.
1. Slides with the blood or bone marrow smear 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 J.lI of RNase solution/slide
and cover with a 24x50 mm coverslip [RNase solution: per slide 100 J.lI
2xSSC plus 1 J.lI of RNase stock (= 5 J.lg/J.l1)].
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment 2).
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 itwith 100ml1 xPBS (RT)for5min (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 2).
(Pepsin-buffer: Add 1 ml of 1 M Hel to 99 ml of distilled water and
incubate at 37°C for about 20 min; then add 50 J.lI 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/MgCh, incubate at RT for 5 min with
gentle agitation. MgCh will block the enzymatic activity of pepsin
(lxPBS/MgCh = 5% (v/v) 1M MgCh in 1xPBS).
8. PostfIx nuclei on the slide surfaces by replacing 1xPBS/MgCh 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 xPBS 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 3).
THOMAS LIEHR
Slide pretreatment
During the whole slide pretreatment procedure, the slides lose the red
erythrocytes and they become more and more transparent. At the end
(step 10), the nuclei of the blasts and lymphocytes are the only remaining
cellular components on the slide surface. If the cellular structure is to be
maintained and tumor cells are to be distinguished from nontumor cells,
this slide pretreatment step must be replaced by a lO-min incubation of
the slides in 2x SSC at room temperature, prior to the FISH.
1. Slides with the blood or bone marrow smear 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 J.lI of RNase solution/slide
and cover with a 24x50 mm coverslip [RNase solution: per slide 100 J.lI
2xSSC plus 1 J.lI of RNase stock (= 5 J.lg/J.l1)].
3. Incubate the slides in a humid chamber for 15 min at 37°C (see comment 2).
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 itwith 100ml1 xPBS (RT)for5min (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 2).
(Pepsin-buffer: Add 1 ml of 1 M Hel to 99 ml of distilled water and
incubate at 37°C for about 20 min; then add 50 J.lI 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/MgCh, incubate at RT for 5 min with
gentle agitation. MgCh will block the enzymatic activity of pepsin
(lxPBS/MgCh = 5% (v/v) 1M MgCh in 1xPBS).
8. PostfIx nuclei on the slide surfaces by replacing 1xPBS/MgCh 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 xPBS 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 3).