94
THOMAS LIEHR
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 4xSSCI0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 J.lI of solution I to each slide, cover with 24 x 50 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 4xSSCI0.2% Tween (RT,
with gentle agitation).
14. Add 50 J.lI 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 J.lI of DAPI (4,6-diamidino-2phenylindol 2 HCI stock-solution; Serva 18860) in 100 ml 4xSSC +
0.2% Tween; make fresh as required].
18. Wash slides several times in water for a few seconds and air dry.
19. Add 15 J.lI of antifade Vectashield (CAMON Vector Laboratories
H1000), cover with coverslips and look at the results under a fluorescence microscope.
Comments
1. Sodium citrate-treated blood or bone marrow is best suited for the
preparation of a "smear-slide" as it spreads the best. However, blood
orbone marrow treated with the other anticoagulants can be used, as well.
2. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations
can be too stringent, resulting in clean slides without any remaining
nuclei.
THOMAS LIEHR
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 4xSSCI0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 J.lI of solution I to each slide, cover with 24 x 50 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 4xSSCI0.2% Tween (RT,
with gentle agitation).
14. Add 50 J.lI 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 J.lI of DAPI (4,6-diamidino-2phenylindol 2 HCI stock-solution; Serva 18860) in 100 ml 4xSSC +
0.2% Tween; make fresh as required].
18. Wash slides several times in water for a few seconds and air dry.
19. Add 15 J.lI of antifade Vectashield (CAMON Vector Laboratories
H1000), cover with coverslips and look at the results under a fluorescence microscope.
Comments
1. Sodium citrate-treated blood or bone marrow is best suited for the
preparation of a "smear-slide" as it spreads the best. However, blood
orbone marrow treated with the other anticoagulants can be used, as well.
2. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations
can be too stringent, resulting in clean slides without any remaining
nuclei.