26 FISH on Insect Cells Transfected with Heterologous DNA
327
13. Put the slides in 4xSSC/0.2% Tween (l00 ml, RT), for a few seconds.
14. Add 50 III of solution I to each slide, cover with 24x50 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].
15. Remove the coverslips and wash 3x3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
16. Add 50 III of solution II to each slide, cover with 24x50 mm coverslips
and incubate at 37°C for 45 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/
4xSSC + 0.2% Tween + 5% BSA (l:100); make fresh as required].
17. Repeat step 15.
18. Repeat steps 14 and 15.
19. 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-2phenylindol.2HCl stock-solution; Serva 18860) in 100 ml 4xSSC/
0.2% Tween; make fresh as required).
20. Wash slides several times in water for a few seconds and air dry.
21. Add 15 III of antifade Vectashield (CAMON Vector Laboratories
H1000), cover with coverslips and look at the results under a fluorescence microscope.
Comments
1. Remember to prepare the cells to be tested for transfection and the wild
type cells as negative controls. The approach described here always
needs a negative control.
2. Incubation in formalin-buffer can be done with or without agitation, it
leads to no difference in the quality of prepared Schneider cells.
3. For the precipitation, sodium acetate does not need to be added; the
dextran blue from the "stopmix" can replace it here.
4. During the washing steps, it is important to prevent the slide surface
drying out, otherwise background problems may arise.
327
13. Put the slides in 4xSSC/0.2% Tween (l00 ml, RT), for a few seconds.
14. Add 50 III of solution I to each slide, cover with 24x50 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].
15. Remove the coverslips and wash 3x3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
16. Add 50 III of solution II to each slide, cover with 24x50 mm coverslips
and incubate at 37°C for 45 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/
4xSSC + 0.2% Tween + 5% BSA (l:100); make fresh as required].
17. Repeat step 15.
18. Repeat steps 14 and 15.
19. 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-2phenylindol.2HCl stock-solution; Serva 18860) in 100 ml 4xSSC/
0.2% Tween; make fresh as required).
20. Wash slides several times in water for a few seconds and air dry.
21. Add 15 III of antifade Vectashield (CAMON Vector Laboratories
H1000), cover with coverslips and look at the results under a fluorescence microscope.
Comments
1. Remember to prepare the cells to be tested for transfection and the wild
type cells as negative controls. The approach described here always
needs a negative control.
2. Incubation in formalin-buffer can be done with or without agitation, it
leads to no difference in the quality of prepared Schneider cells.
3. For the precipitation, sodium acetate does not need to be added; the
dextran blue from the "stopmix" can replace it here.
4. During the washing steps, it is important to prevent the slide surface
drying out, otherwise background problems may arise.