Preparation of Agarose Pads and Glue Down Techniques

Transgenic worms with cell specific pHluorin expression are immobilizing for viewing under perfusion by gluing them onto an agarose pad, as described for Ca2+ imaging using the cameleon biosensor (30). The pads are generally formed from 2% agarose in M9 media; it is essential that water alone not be used, as the divalent cations in the pad accelerate polymerization of the cyanoacrylate adhesive. In addition, the tonicity of the final agarose preparation is important, and should mimic the tonicity of the superfusate to avoid osmotic imbalance and salt-related effects such as swelling or shrinkage of the worm. Depending on the physiological function to be assayed, we will place well-fed worms on an unseeded plate for 1 h prior to mounting them to allow expulsion of the immediate contents of the intestinal lumen.

3.2.1. Pouring the Agarose Pad

1. Melt 1 g of agarose in 50 mL of M9 solution. Allow the agarose to cool slightly. It may be convenient to maintain the melted agarose in a heating block at 65°C for later use.

2. Place approx 8 ^L of the melted agarose onto clean 50-mm cover glass (0.17 mm optical quality). Working quickly, before the agarose hardens, place a second 40mm cover glass on top of the first to form a small, thin patch. Avoid bubbles. Slide this patch gently using the top piece of cover glass so that it will be positioned in the center of the viewing area when the 50-mm cover glass is mounted to form the bottom of the perfusion chamber.

3. Pry the top cover glass off the pad, taking care that the pad remains firmly attached to the bottom cover glass. Then, use a small, flame-drawn capillary to quickly apply a thin coating of Nexaband S/C cyanoacrylate glue to the edges of the pad. This will prevent the pad from floating upward during perfusion.

3.2.2. Gluing the Worm to the Pad

1. Working quickly, use a worm pick to move a small amount of bacterial lawn to the agarose (to encourage the worm to crawl off the pick), then move the worm itself onto the pad. Take special care not to break through the pad with the pick during the transfer, as this will result in the pad lifting during imaging. A longer, slightly rounded pick will work better than a wide, flat pick.

2. Apply a small amount of Nexaband S/C veterinary adhesive to the worm using a patch pipet or flame drawn capillary with the tip broken off. The adhesive polymerizes upon contact with divalent cations in the pad, and is ideal for immobilizing worms for viewing under superfusate over a prolonged time period. Use the smallest amount of adhesive that results in immobilization, as excess adhesive will dehydrate the worm. The butyl derivative appears to be more biocompatible and, to avoid autofluorescence, the clear adhesive should be used. The slightest touch of the capillary to the worm should result in enough adhesive to prevent motion. More extensive contact may allow polymerization of the adhesive within the capillary itself.

3. Immediately place a small droplet of superfusate on the worm to prevent dehydration. Serotonin can be included at 2 mM to stimulate pharyngeal pumping, as necessary. Use the cover glass to form the bottom of a perfusion chamber, with the worm centered and facing upward. A small amount of silicone grease applied to the bottom of the chamber will help form a seal. The chamber is then moved to a mounting platform, where it is secured and placed onto a microscope.

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