182
BERND RAUTENSTRAUSS and THOMAS LIEHR
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 !JI 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 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!JI of solution II to each slide, cover with 24x50 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)1 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 !JI of DAPI (4,6-diamidino-2phenylindo1.2HCI stock-solution; Serva 18860) in 100 ml 4xSSCI
0.2% Tween; make fresh as required].
19. Wash slides several times in water for a few seconds and air dry.
20. Add 15 !JI of antifade Vectashield (CAMON Vector Laboratories
HIOOO), cover with coverslips and look at the results under a fluorescence microscope.
Comments
1. If only small amounts of cryoftxed tissue are available, tissue can be cut
on a cryostat. About 15-25 !Jm cryosections (2-5 mm in diameter) are
sufficient for about 5 slides with extracted interphase nuclei.
2. Incubation of the tissue in formalin buffer can be done by agitation as
well, with no difference in the quality or quantity of resulting extracted
nuclei.
3. If only small amounts of tissue are to be digested (see comment 1) 0.2004 ml of PK-solution are sufficient.
BERND RAUTENSTRAUSS and THOMAS LIEHR
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 !JI 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 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!JI of solution II to each slide, cover with 24x50 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)1 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 !JI of DAPI (4,6-diamidino-2phenylindo1.2HCI stock-solution; Serva 18860) in 100 ml 4xSSCI
0.2% Tween; make fresh as required].
19. Wash slides several times in water for a few seconds and air dry.
20. Add 15 !JI of antifade Vectashield (CAMON Vector Laboratories
HIOOO), cover with coverslips and look at the results under a fluorescence microscope.
Comments
1. If only small amounts of cryoftxed tissue are available, tissue can be cut
on a cryostat. About 15-25 !Jm cryosections (2-5 mm in diameter) are
sufficient for about 5 slides with extracted interphase nuclei.
2. Incubation of the tissue in formalin buffer can be done by agitation as
well, with no difference in the quality or quantity of resulting extracted
nuclei.
3. If only small amounts of tissue are to be digested (see comment 1) 0.2004 ml of PK-solution are sufficient.