1. Mount the slide with 100 |L of blocking buffer and a cover slip. Leave the slide at room temperature for 5 min.

2. Remove the cover slip, drain blocking buffer from the slide, and apply 100 ||L of a detection solution mix (see Note 10) to the slide.

3. Place a cover slip and incubate the slide in a moist chamber at 37°C for 30 min.

4. Remove the cover slip and wash the slide three times for 5 min each in washing buffer at room temperature with gentle agitation.

5. As soon as the slide has air-dried in the dark, apply 10 to 20 |L of DAPI counter-staining solution to the slide and cover the slide with a cover slip.

6. Examine the slide under the fluorescence microscope and image system.

4. Notes

1. The lyophilized oligonucleotide is stable at -20°C for 1 yr or longer. It is generally accepted that oligonucleotides dissolved in TE are stable for at least 6 mo at -20°C or 4°C. Oligonucleotides dissolved in water are stable for at least 6 mo at -20°C. Do not store oligonucleotides in water at 4°C. TE is recommended compared with deionized water because the pH value of the water is often slightly acidic and can cause oligonucleotide hydrolysis.

2. In our experience, the MJ Research Thermocycler (PTC-100) gives us the best results. The Hybrid from Vysis Inc can be used, but the temperature program variation should be validated to perform multi-PRINS adequately.

3. In the detection solution, a relatively weak fluorochrome, for example, avidin-fluorescein or anti-dig-fluorescein (green), should be used for the last PRINS target labeling to minimize the color mixing in the previous PRINS target. Whereas the first labeling uses biotin-dUTP and the second labeling uses dig-dUTP (bio-dig) for two different chromosome targets, respectively, an appropriate fluorochrome mix should be avidin-rhodamine/anti-dig-fluorescein. Conversely, if the labeling order is dig-bio, the fluorochrome mix should be anti-dig-rhodamine/avidin-fluorescein. The principle of selecting a relatively weak fluorochrome for the last PRINS target detection is critical for double PRINS. For the triple PRINS reaction, the weak fluorochrome should be used to detect the first and last PRINS targets.

4. The efficiency of the PRINS relies greatly on the temperature conditions and the humidity when preparing slides. The optimal conditions for the dropping of cells onto slides can be reached by using a Thermotron (CDS-5) (16) or in a temperature/humidity-adjustable chamber. The best conditions for PRINS may vary from laboratory to laboratory and should be determined by testing beforehand. Ideally, the nuclei on the slide should show a gray color, and no reflective nuclei or bright rings around any nuclei should be observed.

5. The PRINS reaction works optimally with freshly prepared slides. The slides should be completely dry and are then ready for PRINS. However, slides stored at -20°C for up to 1 mo can be used for the PRINS reaction.

6. For uncultured amniotic cells, necessary pretreatment is performed by incubating the slides in formaldehyde-PBS/MgCl2 solution (100 mL PBS/50 mMMgCl2 and 2.7 mL 37% formaldehyde) for 1 min at room temperature, followed by rinsing in 1X PBD buffer (0.1 M NaH2P04/0.1 M Na2HPO4 and 0.1% Nonidet) for 1 min before the dehydration step using an ethanol series.

7. 5% Glycerol can effectively prevent the drying of the PRINS reaction solution during the annealing and extension steps of PRINS on the heating block of the themocycler. Higher concentrations of glycerol may cause inactivation of DNA polymerase.

8. A single step of primer annealing and strand extension for most primers was performed at 62.5°C for 15 min. For X chromosome detection, the PRINS program is as follows: annealing at 65°C for 10 min and extension to 72°C for 10 min. For the Y chromosome, annealing is at 56°C for 10 min and extension is at 72°C for 10 min.

9. Washing at room temperature for 5 min usually is sufficient. In the case of high background interference, the slide can be washed once at 45°C for 5 min and twice at room temperature for 5 min.

10. When the labeling order is bio-dig-bio, a 100-pL mix of 1% avidin-fluorescein DCS and 1% anti-dig-rhodamine is used; however, when the labeling order is dig-bio-dig, a 100-pL mix of 1% anti-dig-fluorescein and 1% avidin-rhodamine is used. In triple PRINS, a new color, yellow, is created for the first PRINS target because it mixes the green labeling twice and the red labeling once (Fig. 1).

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