Slide Preparation and Conservation

1. Harvesting of the cells and spreading: in our experiments, the cells were harvested by centrifugation at 200^ for 10 min and suspended in cell suspension medium. Then, they were cytocentrifuged on cover slips (24 X 36 mm) for 4 min at 500^ using a cytospin (see Note 6).

2. Immediately fix the cells in a 90% methanol/10% acetic solution during 4 min and then rinse and rehydrate for 5 min in 1X PBS (see Note 3).

3. Stockade: At this step, cells can be used or allowed to dry before frozen for stockade in hermetic boxes with a desiccator-like silica gel (see Note 7). To use some of the frozen cover slips, warm the box to 37°C for 30 min before opening. The remaining slides could be frozen again without damage. Start the experiment at the rehydratation stage.

4. Permeabilize the rehydrated slides in permeabilization medium (0.1% Triton in 1X PBS, see Note 8) for 7 min. Then, dehydrate in 70%, 85%, and 100% ethanol whashes (4 mn each) and allow to dry.

Fig. 1. The PRINS-IFF methodology.

5. Denature the slides in the denaturation solution at 71°C for 2 min and then stop them in a series of cold ethanol washes maintained at 4°C in ice (see Note 9): 70%, 80%, 90%, and 100%, 2 min each, respectively, except for the last, during which the slides are allowed to stay for 4 min. After drying the slides at room temperature, immediately perform the PRINS reaction (see Note 10).

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