Preparation of Samples

1. Drop the cell suspension onto precleaned slides. Thermotron conditions should be 25°C and 50% humidity (see Notes 4 and 5).

2. Incubate the slides in 2X SSC for 15 to 30 min at 37°C and then dehydrate the slides through a series of ethanol (70%, 80%, and 100%) at room temperature. For amniotic cells, a specific pretreatment is applied (see Note 6).

3. Prepare 50 mL of denaturation solution (70% formamide/2X SSC) by mixing thoroughly 35 mL of formamide (ultrapure grade, Fluka), 5 mL of 20X SSC, and 10 mL of distilled water in a glass Coplin jar at a pH value of 7.2-7.4. Before use, prewarm the solution until it reaches 70°C. Between uses, store denaturation solution at 2 to 8°C and discard it after 3 to 5 d.

4. Denature slides in 70% formamide/2X SSC at 70°C for 2 min and successively pass them through 70%, 80%, and 100% ethanol for 2 min each at -20°C. After air-drying, the slides are ready for the PRINS procedure.

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