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SUSANN NEUBAUER
10. Put the 35 III of the denaturated and preannealed probe (point 6) onto
the denatured slide, cover with a coverslip (24x50 mm) without making bubbles and seal with rubber cement.
11. Transfer the slides for hybridization in a humid chamber at 37°C for
2 nights.
12. Remove the rubber cement and place the slides in a Coplin jar filled
with 4xSSC/0.2%Tween to remove the coverslips carefully.
13. Post-wash the slides in prewarmed 4xSSC solution containing 50%
formamide (45°C) and in 0.1 xSSC (60°C) 3x5 min each.
14. Immerse the slides in a Coplin jar with 4xSSC/0.2% Tween.
15. Add 150 III 5% BSA blocking solution (bovine serum albumin (Sigma)
in 4xSSC/0.2% Tween) onto each slide, cover with a coverslip 24x60
mm and incubate for 20 min at 37°C in a humid chamber.
16. Remove the coverslips and place the slides in a Coplin jar with 4x SSC/
0.2% Tween.
17. Just before use, prepare detection solution I and pipette 100 III onto
each slide and incubate at 37°C for 60 min in a humid chamber (detection solution I: FIrC-avidin (CAMON Vector Laboratories A2011) diluted 1:500 in 5%BSA solution).
18. Remove the coverslips and wash 3x5 min with 4xSSC/0.2% Tween at
37°C.
19. Add 100 III of detection solution II to each slide, cover with a coverslip
and incubate in a humid chamber at 37°C for 2 h (prepare fresh detection solution II: biotinylated antiavidin [CAMON Vector Laboratories
BA0300] diluted 1:100 and Anti-digoxigenin-rhodamine diluted 1:35
[Boehringer Mannheim, 1207750] in 5%BSA solution).
20. Repeat step 16.
21. Add 100 III detection solution I on each slide, cover with a coverslip and
incubate in a humid chamber at 37°C for 60 min and repeat step 16.
22. Counterstaining is performed with 4,6-diamidinophenylindol (DAPI)
[1 Ilg dissolved in 1 ml 4xSSC/Tween]; incubate 5 min in the dark.
23. Immerse the slides 3-4 times in distilled water, air dry, add 20 III antifade Vectashild and cover with a 24x60 mm coverslip.
SUSANN NEUBAUER
10. Put the 35 III of the denaturated and preannealed probe (point 6) onto
the denatured slide, cover with a coverslip (24x50 mm) without making bubbles and seal with rubber cement.
11. Transfer the slides for hybridization in a humid chamber at 37°C for
2 nights.
12. Remove the rubber cement and place the slides in a Coplin jar filled
with 4xSSC/0.2%Tween to remove the coverslips carefully.
13. Post-wash the slides in prewarmed 4xSSC solution containing 50%
formamide (45°C) and in 0.1 xSSC (60°C) 3x5 min each.
14. Immerse the slides in a Coplin jar with 4xSSC/0.2% Tween.
15. Add 150 III 5% BSA blocking solution (bovine serum albumin (Sigma)
in 4xSSC/0.2% Tween) onto each slide, cover with a coverslip 24x60
mm and incubate for 20 min at 37°C in a humid chamber.
16. Remove the coverslips and place the slides in a Coplin jar with 4x SSC/
0.2% Tween.
17. Just before use, prepare detection solution I and pipette 100 III onto
each slide and incubate at 37°C for 60 min in a humid chamber (detection solution I: FIrC-avidin (CAMON Vector Laboratories A2011) diluted 1:500 in 5%BSA solution).
18. Remove the coverslips and wash 3x5 min with 4xSSC/0.2% Tween at
37°C.
19. Add 100 III of detection solution II to each slide, cover with a coverslip
and incubate in a humid chamber at 37°C for 2 h (prepare fresh detection solution II: biotinylated antiavidin [CAMON Vector Laboratories
BA0300] diluted 1:100 and Anti-digoxigenin-rhodamine diluted 1:35
[Boehringer Mannheim, 1207750] in 5%BSA solution).
20. Repeat step 16.
21. Add 100 III detection solution I on each slide, cover with a coverslip and
incubate in a humid chamber at 37°C for 60 min and repeat step 16.
22. Counterstaining is performed with 4,6-diamidinophenylindol (DAPI)
[1 Ilg dissolved in 1 ml 4xSSC/Tween]; incubate 5 min in the dark.
23. Immerse the slides 3-4 times in distilled water, air dry, add 20 III antifade Vectashild and cover with a 24x60 mm coverslip.