PRINS Reaction

1. Prepare the PRINS reactional mixture (14): for a mix of 50 ||L (x number of cover slips + one): 42.5 ||L of distillated water, 5 |L of Taq polymerase buffer (with MgCl2), 0.5 |L of dNTPs mix, 0.5 |L of fluorescein-12-dUTP, 1 |L of primers (100 pM) and 0.5 ||L (2 units) of Taq polymerase. Maintain the mix on ice and shield it from the light (see Note 11).

2. Perform PRINS reactions on a programmable thermal cycler equipped with a flat plate block (see Notes 10 and 12). The PRINS reaction consists of two programmed steps: 15 min corresponding to the specific annealing temperature of the primers (55°C in our case), followed by 15 min at 72°C to allow the in situ chain elongation (see Note 13). Only one cycle is realized. Slides corresponding to the number of cover slips are put on the block and the cycle is initiated. As soon as the annealing temperature is reached, deposit 50 |L of the PRINS reactional mix on each slide. To prevent bubbles, the denatured cover slips should be placed carefully on the slides (the cells between the cover slip and the slide) and sealed using rubber cement.

3. At the end of the reaction, remove the rubber cement and the wash cover slips three times in 4X SSC washing solution. Then, dehydrate cover slips in an ethanol series (70%, 85%, and 100%) for 3 min each, and immediately start the immunofluorescence procedure (see Note 10).

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