6 Epithelial Cells from Buccal Smears and Urine
103
2. Remove coverslip and wash 3x 3 min in 2x SSC at room temperature in
a Coplin jar on a shaker.
3. Add 50 III of pepsin stock solution to a Coplin jar with 100 ml in a 37°C Pepsin
water bath and incubate the slide for 10 min in the Pepsin solution.
4. Wash 1 x 5 min in IxPBS in a Coplin jar on a shaker at room temperature.
5. Wash 5 min in PBS-MgCh.
6. Wash 10 min in 1% formaldehyde in PBS-MgCl z .
7. Wash for 5 min in PBS.
8. Dehydrate in a series of ethanol (70%, 90%, 100%) for 2 min each.
9. Air dry slides.
Denaturation and hybridization
1. Cover slide with 100 III 70% formamide/2 x SSC and a coverslip and
incubate it for 5 min on the back of a metal box in a 75°C water
bath for denaturation of target DNA.
2. Dehydrate slide in a series of ethanol (70%, 90%,100%) on ice for 2 min
each to avoid rehybridization.
3. While slide is air drying, denaturate specific DNA probe in 10 III hybridization mix for 5 min at 75°C and place on ice immediately.
4. Place denatured probe (10 Ill) on one part of the slide, cover carefully
with a 20x20 mm coverslip avoiding air bubbles and seal with rubber
cement.
5. Incubate in a moist chamber at 37°C over night or at least 4 h for centromeric probes.
Posthybridization washes
For commercially available probes, follow the instructions of the manufacturers or:
103
2. Remove coverslip and wash 3x 3 min in 2x SSC at room temperature in
a Coplin jar on a shaker.
3. Add 50 III of pepsin stock solution to a Coplin jar with 100 ml in a 37°C Pepsin
water bath and incubate the slide for 10 min in the Pepsin solution.
4. Wash 1 x 5 min in IxPBS in a Coplin jar on a shaker at room temperature.
5. Wash 5 min in PBS-MgCh.
6. Wash 10 min in 1% formaldehyde in PBS-MgCl z .
7. Wash for 5 min in PBS.
8. Dehydrate in a series of ethanol (70%, 90%, 100%) for 2 min each.
9. Air dry slides.
Denaturation and hybridization
1. Cover slide with 100 III 70% formamide/2 x SSC and a coverslip and
incubate it for 5 min on the back of a metal box in a 75°C water
bath for denaturation of target DNA.
2. Dehydrate slide in a series of ethanol (70%, 90%,100%) on ice for 2 min
each to avoid rehybridization.
3. While slide is air drying, denaturate specific DNA probe in 10 III hybridization mix for 5 min at 75°C and place on ice immediately.
4. Place denatured probe (10 Ill) on one part of the slide, cover carefully
with a 20x20 mm coverslip avoiding air bubbles and seal with rubber
cement.
5. Incubate in a moist chamber at 37°C over night or at least 4 h for centromeric probes.
Posthybridization washes
For commercially available probes, follow the instructions of the manufacturers or: