6 Epithelial Cells from Buccal Smears and Urine
101
- Commercially available, directly labeled probes should be used with Hybridization mix
provided hybridization mix following supplier's instructions.
- Self-labeled probes (e.g. with Roche Corp. Nick Translation Mix according to supplier's instructions) use with 20% dextran sulfate: dissolve 2 g dextran sulphate in 5 ml deionized formamide plus 1 ml phosphate buffer plus 1 ml 20xSSC plus 3 ml aqua bidest, store at -20°e.
- 1xSSC: add 25 ml20xSSC to 475 ml aqua bidest, adjust pH to 7.0-7.5 Washing solutions
with 5 drops of 1 M HCl. Store at room temperature
- 2x SSC/O.l % NP-40: add 50 ml20 x SSC to 450 ml aqua bidest, ad 0.5 ml
NP-40, adjust pH to 7.0-7.5 with 1 drop of HCI 32%
- Dapi-solution: per slide, mix 1 ml 2xSSC/0.l% NP-40 with I III Dapi Counterstaining
stock solution (plus 1 III propidium iodide stock solution if there is not
a red fluorochrome being used to detect DNA probe)
- Dapi stock solution: dissolve 10 mg DAPI (Serra) in 10 ml aqua dest
and store aliquots of 50 III at -20°C (the aliquot at use can be stored at
4°C)
- Propidium iodide stock solution: dissolve 10 mg propidium iodide
(Sigma) in 10 ml aqua bidest and store aliquots of 50 III at -20°C
(the aliquot at use can be stored at 4°C)
- Antifade: Mounting medium for fluorescence (Vectashield)
Procedure
Preparation of buccal smears
1. Scrape the inner surface ofthe buccal mucosa and saliva with a wooden
tongue depressor and smear it onto a slide.
2. Let the slide air dry.
3. Transfer the slide into a Quadriperm tray.
4. Carefully overlay the slide with Otto's solution and incubate for 30-60
min at 37°e.
5. Remove Otto's solution carefully and overlay the slide with fixative,
incubate at room temperature for 5 min.
101
- Commercially available, directly labeled probes should be used with Hybridization mix
provided hybridization mix following supplier's instructions.
- Self-labeled probes (e.g. with Roche Corp. Nick Translation Mix according to supplier's instructions) use with 20% dextran sulfate: dissolve 2 g dextran sulphate in 5 ml deionized formamide plus 1 ml phosphate buffer plus 1 ml 20xSSC plus 3 ml aqua bidest, store at -20°e.
- 1xSSC: add 25 ml20xSSC to 475 ml aqua bidest, adjust pH to 7.0-7.5 Washing solutions
with 5 drops of 1 M HCl. Store at room temperature
- 2x SSC/O.l % NP-40: add 50 ml20 x SSC to 450 ml aqua bidest, ad 0.5 ml
NP-40, adjust pH to 7.0-7.5 with 1 drop of HCI 32%
- Dapi-solution: per slide, mix 1 ml 2xSSC/0.l% NP-40 with I III Dapi Counterstaining
stock solution (plus 1 III propidium iodide stock solution if there is not
a red fluorochrome being used to detect DNA probe)
- Dapi stock solution: dissolve 10 mg DAPI (Serra) in 10 ml aqua dest
and store aliquots of 50 III at -20°C (the aliquot at use can be stored at
4°C)
- Propidium iodide stock solution: dissolve 10 mg propidium iodide
(Sigma) in 10 ml aqua bidest and store aliquots of 50 III at -20°C
(the aliquot at use can be stored at 4°C)
- Antifade: Mounting medium for fluorescence (Vectashield)
Procedure
Preparation of buccal smears
1. Scrape the inner surface ofthe buccal mucosa and saliva with a wooden
tongue depressor and smear it onto a slide.
2. Let the slide air dry.
3. Transfer the slide into a Quadriperm tray.
4. Carefully overlay the slide with Otto's solution and incubate for 30-60
min at 37°e.
5. Remove Otto's solution carefully and overlay the slide with fixative,
incubate at room temperature for 5 min.