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14. Let the slide air dry.
15. If not fIxed at step 13, transfer the slide into a Quadriperm tray.
16. Overlay the slide with fIxative, incubate at room temperature for 5 min.
17. Remove fIxative and incubate another 5 min at room temperature.
18. Remove fIxative once again and incubate another 5 min at room temperature.
19. Remove fIxative and let the slide air dry.
20. If the slide is not being used within a few days store it at -20°C.
RNase/pepsin pretreatment
RNase 1.
2.
Pepsin 3.
4.
5.
6.
7.
8.
9.
Cover slide with 100 III RNase working solution and a coverslip and
incubate in a metal tray in a 37°C water bath for 3 min.
Remove coverslip and wash 3x 3 min in 2x SSC at room temperature in
a Coplin jar on a shaker.
Add 50 III of pepsin stock solution to a Coplin jar with 100 ml in a 37°C
water bath and incubate the slide for 8 min in the pepsin solution.
Wash 1 x 5 min in 1x PBS in a Coplin jar on a shaker at room temperature.
Wash 5 min in PBS-MgCl z .
Wash 10 min in 1% formaldehyde in PBS-MgClz.
Wash for 5 min in PBS.
Dehydrate in a series of ethanol (70%, 90%, 100%) for 2 min each.
Air dry slides.
Denaturation and hybridization
1. Cover slide with 100 III 70% formamide/2 x SSC and a coverslip and
incubate it for 6 min on the back of a metal box in an 86°C water
bath for denaturation of the target DNA.
2. Dehydrate slide in a series of ethanol (70%,90%, 100%) on ice for 2 min
each to avoid rehybridization.