7 FISH on Hair Root Cells
113
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 J..lI 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].
13. Remove the coverslips and wash 3x3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
14. Add 50 J..lI of solution II to each slide, cover with 24 x 50 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 steps 12 and 13.
17. Counterstain the slides with DAPI-solution (100 ml in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 5 J..lI of DAPI (4,6-diamidino-2phenylindol. 2HCI 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 f.ll of antifade Vectashield (CAMON Vector Laboratories
HI000), cover with coverslips and look at the results under a fluorescence microscope.
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 nuclei.
2. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. If longer storage is necessary, slides are stable at -20°C
for several months.
3. In other FISH-protocols with denaturation times of 2-3 min only, the
maintenance of available metaphase chromosomes is the main aspect,
which is of no significance in the actual protocol.
113
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 J..lI 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].
13. Remove the coverslips and wash 3x3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
14. Add 50 J..lI of solution II to each slide, cover with 24 x 50 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 steps 12 and 13.
17. Counterstain the slides with DAPI-solution (100 ml in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 5 J..lI of DAPI (4,6-diamidino-2phenylindol. 2HCI 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 f.ll of antifade Vectashield (CAMON Vector Laboratories
HI000), cover with coverslips and look at the results under a fluorescence microscope.
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 nuclei.
2. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. If longer storage is necessary, slides are stable at -20°C
for several months.
3. In other FISH-protocols with denaturation times of 2-3 min only, the
maintenance of available metaphase chromosomes is the main aspect,
which is of no significance in the actual protocol.