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BERND RAUTENSTRAUSS and CHRISTINA FUCHS
9. Take the slides out of 37°C, remove rubber cement with forceps and
cover slips by letting them swim off in 4xSSC/0.2%Tween (RT, 100 ml
Coplin jar).
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/0.2% Tween (l00 ml, RT), for a few seconds.
12. Add 50 III of solution 1 to each slide, cover with 24 x 50 mm coverslips
and incubate at 37°C for 30 min in a humid chamber. [Solution 1:
FITC-avidin (CAMON Vector Laboratories A2011)/4xSSC/0.2%
Tween/5% BSA (1:300 both Sigma); make fresh as required].
13. Remove the coverslips and wash 3x3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
14. Add 50 III of solution 2 to each slide, cover with 24x50 mm coverslips
and incubate at 37°C for 75 min in a humid chamber. [Solution 2: Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/anti-digoxigenin-rhodamine (Boehringer Mannheim, 1207750)/4xSSC/0.2%
Tween/5% BSA (1:20:100); make fresh as required].
15. Repeat step 13.
16. Repeat step 12.
17. Repeat step 13.
18. Counterstain the slides with DAPI-solution (100 ml in a Coplin jar, RT)
for 8 min. (DAPI-solution: Dissolve 5 III of DAPI (4,6-diamidino-2phenylindo1.2HCl stock-solution; Serva 18860) in 100 ml 4xSSC/
0.2% Tween; make fresh as required).
19. Wash slides several times in deionised water for a few seconds and air
dry.
20. Add 15 III of antifade Vectashield (CAMON Vector Laboratories
HI000), cover with cover slips and look at the results under a fluorescence microscope.