4 FISH on Uncultured Blood or Bone Marrow
87
7. Add 20 ~l of probe-solution onto each denatured slide, put 24x50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for three nights at 37°C in a humid chamber (see comment 4).
9. Take the slides out of 37°C, remove rubber cement with forceps and
then the coverslips by letting them swim off in 4xSSC/O.2%Tween (RT,
100-ml Coplin jar) (see comment 5).
10. Postwash the slides 3x 5 min in formamide-solution (45°C) followed
by 3x5 min in 2xSSC (37°C) in a 100-ml Coplin jar, with gentle agitation.
11. Put the slides in 4xSSC/O.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 ~l of solution I to each slide, cover with 24x50 mm coverslips
and incubate at 37°C for 30 min in a humid chamber. [Solution I: FITCavidin (CAMON Vector Laboratories A2011}/4xSSC + 0.2% Tween +
5% BSA (1:300); make fresh as required].
13. Remove the coverslips and wash 3x3 min in 4xSSC/O.2% Tween (RT,
with gentle agitation).
14. Add 50 ~l of solution II to each slide, cover with 24 x 50 mm coverslips
and incubate at 37°C for 75 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/antidigoxigenin-rhodamine (Boehringer Mannheim, 1207750)/4xSSC +
0.2% Tween + 5% BSA (1:20:100); make fresh as required].
15. Repeat step 13.
16. Repeat steps 12 and 13.
17. Counterstain the slides with DAPI-solution (100 ml in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 5 ~l of DAPI (4,6-diamidino-2phenylindo1.2HCI stock-solution; Serva 18860) in 100 ml 4xSSCI
0.2% Tween; make fresh as required].
18. Wash slides several times in water for a few seconds and air dry.
19. Add 15 ~l of antifade Vectashield (CAMON Vector Laboratories
HlOOO), cover with coverslips and look at the results under a fluorescence microscope.
87
7. Add 20 ~l of probe-solution onto each denatured slide, put 24x50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for three nights at 37°C in a humid chamber (see comment 4).
9. Take the slides out of 37°C, remove rubber cement with forceps and
then the coverslips by letting them swim off in 4xSSC/O.2%Tween (RT,
100-ml Coplin jar) (see comment 5).
10. Postwash the slides 3x 5 min in formamide-solution (45°C) followed
by 3x5 min in 2xSSC (37°C) in a 100-ml Coplin jar, with gentle agitation.
11. Put the slides in 4xSSC/O.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 ~l of solution I to each slide, cover with 24x50 mm coverslips
and incubate at 37°C for 30 min in a humid chamber. [Solution I: FITCavidin (CAMON Vector Laboratories A2011}/4xSSC + 0.2% Tween +
5% BSA (1:300); make fresh as required].
13. Remove the coverslips and wash 3x3 min in 4xSSC/O.2% Tween (RT,
with gentle agitation).
14. Add 50 ~l of solution II to each slide, cover with 24 x 50 mm coverslips
and incubate at 37°C for 75 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/antidigoxigenin-rhodamine (Boehringer Mannheim, 1207750)/4xSSC +
0.2% Tween + 5% BSA (1:20:100); make fresh as required].
15. Repeat step 13.
16. Repeat steps 12 and 13.
17. Counterstain the slides with DAPI-solution (100 ml in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 5 ~l of DAPI (4,6-diamidino-2phenylindo1.2HCI stock-solution; Serva 18860) in 100 ml 4xSSCI
0.2% Tween; make fresh as required].
18. Wash slides several times in water for a few seconds and air dry.
19. Add 15 ~l of antifade Vectashield (CAMON Vector Laboratories
HlOOO), cover with coverslips and look at the results under a fluorescence microscope.