Construction of the Culture Chambers

We use culture chambers similar to those described by Nicklas and Staehly (22). Ours consist of an aluminum slide (instead of a glass slide) with a 20-mm-diameter (instead of 15-mm) hole covered on one side with a scrupulously clean (see Subheading 3.1.1.) square cover slip (24 x 24 mm2) that is fixed to the slide with nail polish (instead of petroleum jelly).

3.1.1. Cleaning the Cover Slips

Cover slips may have a thin film of oil to keep them dust-free. This must be removed to reliably maintain living cells on them. Cover slips have a tendency to stack up, so care must be taken to ensure that all surfaces are exposed during the cleaning process.

1. Place the cover slips in the porcelain racks. Always use forceps to manipulate the cover slips (see Note 1).

2. Place the racks into Pyrex beakers filled with 1 L cleaning solution 7X® PF 1%. Use long forceps to manipulate the trays (see Note 2).

3. Boil everything for 10 min in a microwave oven, taking care that the soapy solution does not overflow while boiling. This step will remove the film protecting the cover slips.

4. Let the cover slips cool down at room temperature for 5 min and rinse them with abundant distilled water, being careful not to move the cover slips away from the racks.

5. Fill the Pyrex beaker again with distilled water and repeat step 3. This will remove the remaining soap from the glass surface. Repeat this step until the soap has completely disappeared (at least twice).

6. Cool the cover slips down for 5 min and rinse again using ultrapure water.

7. Fill the Pyrex beaker with ultrapure water and boil once more for 10 min. This step will eliminate ions coming from the standard distilled water.

8. Repeat step 6.

9. Place the porcelain racks carrying the cover slips on a filter paper to drain the excess of water. Use a clean tissue to absorb the water from the edge of the cover slips, without touching the surface of the glass.

10. Put the cover slips together with the racks into a glass box filled with absolute ethanol and store it closed at room temperature.

3.1.2. Mounting the Culture Chambers

1. Keep the aluminum slide in acetone for 5 min to remove any adhered particles, and let it dry.

2. Place it horizontally on the slide's support in such a way that only the two extremes of the slide make any contact.

3. Take a treated cover slip from the alcohol using curved forceps, drain the edges on a tissue, and burn the remaining alcohol in a bunsen burner.

4. Place the cover slip on the slide, covering the well, and seal the edges with nail polish.

5. Store the culture chambers inside the slide's support, keeping the open side of the hole facing down.

6. Let the culture chambers dry for at least 30 min before use.

3.1.3. Cleaning and Recycling the Slides

1. Place the used slides in a removable staining tray and immerse the tray in a staining glass box filled with acetone to remove the attached cover slip.

2. Transfer the tray with the slides through three staining glass boxes filled with xylene, keeping them for at least 4 h in each one. This will remove the oil from the slides.

3. Transfer the tray through three other glass boxes filled with alcohol, keeping the slides in each for at least 4 h. This will remove any xylene residues.

4. Put the tray inside a dry glass box and store it closed. The slides are now ready to be reused starting at step 1 of Subheading 3.1.2.

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