PRINS Reaction

1. Denature the samples by immersing them in the formamide solution (see Note 4) at 72°C for 2 min and then dehydrate them in a series (70%, 90%, and 100%) of ice-cold ethanol washes (4°C) before allowing them to air-dry.

2. Cover each sample with 50 pL of PRINS-modified mixture and top with a cover slide, which is sealed with rubber cement glue. Denature the target DNA at 95°C for 5 min using a hot plate or a thermostat protein-free water bath.

3. Transfer the slides in a hot plate or in a prewarmed water bath at 58°C for 20 min (see Note 13).

4. To perform the elongation step, transfer the slides in a hot plate or in a prewarmed water bath at 72°C for 40 min (see Note 13).

5. Remove the cover slide.

6. Stop the hybridization/elongation reaction by washing the slides with the stop solution containing NaCl and EDTA for 2 min at 60°C.

7. Wash twice with the washing buffer for 5 min per wash at room temperature to remove excess reaction mixture. Place the slides in an orbital shaker and shake during the washing.

8. Place the slides in a blocking solution for 10 min at room temperature to minimize nonspecific signals.

9. Incubate the slides with a solution containing fluorescein-isothiocyanate (FITC)-conjugated antidigoxigenin antibody for 30 min at room temperature in a dark humid chamber.

10. Wash the slides twice with washing buffer for 5 min at room temperature to remove excess antibody. Shake during the washes.

11. To stain the unlabeled DNA, incubate the slides with a PI solution for 4 min in the dark room at room temperature and then wash for 2 to 3 min under running water.

12. Allow to air-dry in the dark.

13. Apply 20 pL of antifading solution on cover slide (24 x 24 mm).

14. Cover each slide with a cover slide. For longer storage and to prevent the cover slide from sliding, the edges of the cover slide can be sealed with nail polish and conserved in the dark at -20°C.

15. Observe slides by immunofluorescence or confocal laser microscopy (see Note 14).

16. Record digitally.

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