430
ANGELA NIETZEL et al.
13. Remove the coverslips and wash 3 x 3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
14. Add 50)11 of solution II to each slide, cover with 24x50 mm coverslips
and incubate at 37
G C for 75 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/
4xSSC + 0.2% Tween + 5% BSA (1:100); make fresh as required].
15. Repeat step 13.
16. Repeat steps 12 and 13.
17. Counterstain the slides with DAPI solution (100 m1 in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 1 )11 of DAPI (4,6-diamidino-2phenylindol.2HCl 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 )11 of antifade Vectashield (CAMON Vector Laboratories
HI000), cover with coverslips and look at the results under a fluorescence microscope equipped with a CCD-camera and an image analyzing system; the latter is necessary, as Cy5 is a dye invisible to the human
eye. Moreover, false colors have to be created by the image analyzing
system for a reliable evaluation.
Comments
1. 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 metaphase spreads or nuclei.
2. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. Iflonger storage is necessary slides are stable at -20
G C for
several months.
3. During the washing steps it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.
ANGELA NIETZEL et al.
13. Remove the coverslips and wash 3 x 3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
14. Add 50)11 of solution II to each slide, cover with 24x50 mm coverslips
and incubate at 37
G C for 75 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/
4xSSC + 0.2% Tween + 5% BSA (1:100); make fresh as required].
15. Repeat step 13.
16. Repeat steps 12 and 13.
17. Counterstain the slides with DAPI solution (100 m1 in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 1 )11 of DAPI (4,6-diamidino-2phenylindol.2HCl 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 )11 of antifade Vectashield (CAMON Vector Laboratories
HI000), cover with coverslips and look at the results under a fluorescence microscope equipped with a CCD-camera and an image analyzing system; the latter is necessary, as Cy5 is a dye invisible to the human
eye. Moreover, false colors have to be created by the image analyzing
system for a reliable evaluation.
Comments
1. 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 metaphase spreads or nuclei.
2. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. Iflonger storage is necessary slides are stable at -20
G C for
several months.
3. During the washing steps it is important to prevent the slide surfaces
drying out, otherwise background problems may arise.