Fixation Of Cultured Cells On Slides

1. Briefly spin down suspended or cultured cells of interest and resuspend them in serum-free medium at a density of 107 cells/ml.

2. Transfer 10 to 15 ml of the cells onto a precoated glass slide and allow cells to attach to the slide in a humid chamber for 40 min.

Three to five replicates should be set up for each cell line. Alternatively, cells can be grown directly on coated glass slides or on coverslips.

3. Place the slides into slide racks and carefully immerse them in a glass staining dish containing a sufficient volume of 10% buffered-formalin or 4% PFA fixation solution.

4. Allow fixation to proceed at room temperature.

Note: Fixation is critical for the success of the in situ hybridization procedure. Fixation time varies with cell lines, ranging from 30 min to 10 h. Therefore, the optimal fixation time should be determined empirically for each cell line.

5. Carefully transfer the slide rack to a staining dish filled with 3X PBS and allow to incubate for 4 min to stop fixation.

6. Rinse the slides 3 x 4 min with 1X PBS in fresh staining dishes.

7. Carry out complete dehydration by transferring the slide rack through a series of 6-min treatments in ethanol dishes (50%, 70%, 95%, 100%, 100%, and 100%).

8. Completely dry the slides at room temperature and store at -80°C in a box containing desiccant until use.

Note: The slides must be completely dried prior to being placed in a freezer in order to prevent potential artifacts during freezing.

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