Preparation and Microinjection of Xenopus Oocytes

Because detailed protocols for preparing and microinjecting Xenopus oocytes are listed elsewhere in this book, we only give a brief summary covering the essential steps. The subheadings outline terminal and survival surgery; removal of epithelial layers, follicular cells, and the vitelline membrane; and oocyte microinjection.

3.1.1. Terminal and Survival Surgery

1. Place a female X. laevis frog (Fig. 1A) in a 0.17% solution of MS-222 (made up in water) until sufficiently anesthetized.

2. If all of the oocytes are to be used, terminal surgery is performed: the frog is anesthetized and decapitated prior to removal of ovaries.

3. If only a few lobes are required for experiments, survival surgery may be performed. Make a small incision (~5-10 mm long) into the abdominal cavity of a fully anesthetized frog and remove a few ovarian lobes (using presterilized instruments) and place them directly into MBS. Suture the incision, using separate stitches for the abdominal wall and skin. Allow the frog to recover in fresh water and monitor for a few hours before returning to the vivarium. Perform alternate surgeries on opposite sides of the frog, with intervals of longer than 1 mo between successive surgeries.

3.1.2. Removal of Epithelial Layers and Follicular Cells

1. Place individual ovarian lobes in MBS, cut them open, and view them under a stereomi-croscope (Fig. 1B).

2. Carefully remove epithelial layers (5) from healthy (uniform pigmentation) stages V and VI oocytes (6) using two fine forceps (Dumont 5): one to hold the ovary membrane steady and the other to manually "peel" the epithelial layers from the oocyte. Repeat this procedure until enough oocytes have been obtained for experiments (typically 200 oocytes).

Fig. 1. (A) Female Xenopus laevis frog. (B) Ovarian lobe (cut open); scale bar = 1 mm. The pigmented (black) and unpigmented hemispheres, termed the animal and vegetal poles, respectively, can be seen.

3. Remove surrounding follicular cells, which are electrically coupled to the oocyte itself through gap junctions. Place oocytes (with the epithelial layers already removed) in approx 5 mL of 0.5 mg/mL collagenase type 1 (in Ringer's solution) with gentle agitation for approx 45 min. Wash repeatedly (about five washes) in MBS.

4. Store oocytes in MBS at 17°C until use. They can be stored in batches of approx 20 oocytes in individual vials filled with about 10 mL MBS. Rinse with fresh MBS twice daily and discard unhealthy oocytes. Alternatively, oocytes can be maintained individually in multiwell plates. Oocytes usually remain healthy for about 7 d. We do not remove the vitelline membrane for standard confocal imaging experiments, but this is stripped (as described in Subheading 3.1.4.) prior to TIRFM or patch-clamp recording.

3.1.3. Microinjection

1. After collagenase treatment, leave oocytes overnight in MBS at 17°C. Oocytes are easier to inject after overnight incubation because the membrane becomes firmer.

2. Pull a glass capillary (Drummond) (Fig. 2A) and examine under the microscope; using a fine pair of forceps, gently break off the end of the needle. For cytoplasmic injections, the needle width can be around 15 to 20 |lm.

3. Backfill the needle with mineral oil, avoiding air bubbles, and assemble it onto the nanoinjector (Fig. 2C). Once the needle is mounted, press the "empty" button on the injector for a few seconds to dispel any air bubbles from the tip.

4. Prepare the solution to be injected. For calcium imaging and photolysis of caged IP3, this is an aqueous solution containing Ca2+ indicator dye (1.5 mM) together with caged IP3 (0.25 mM) d-myo-inositol 1,4,5-trisphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester). For injection of cRNAs, thaw the selected cRNA preparation (e.g., 1/1 mixture of a1B-d and P3 cRNA N-type calcium channel clones) and spin at approx 9000g for 1 min to precipitate any salt crystals.

Fig. 2. Equipment for microinjection. (A) Pipet puller (Narishige, Tokyo, Japan); (B) oocytes aligned for microinjection; and (C) Drummond microinjector.

5. Pipet 1 to 2 ||L of the injection solution onto a piece of fresh Parafilm positioned under the microscope. When injecting cRNA, wear clean gloves and use fresh pipet tips to avoid degradation by RNAases.

6. Using a stereomicroscope, focus on the drop under the microscope and position the micropipet over it; make sure you can see the micropipet enter the drop, then press the "fill" button on the Drummond nanoinjector. After the sample has been loaded, immediately position the tip of the micropipet in the injecting chamber containing MBS to avoid drying solution at the tip.

7. Transfer 10 to 30 oocytes into an injection chamber made by cementing a plastic net into the bottom of a Petri dish to obtain individual "wells" in the bottom of the Petri dish. In this situation, the oocytes can be steadily positioned in each mesh in any required orientation. Line the oocytes up with the equatorial band facing upward toward the injecting needle to avoid penetrating the nucleus (Fig. 2B).

8. Position the micropipet as vertically as possible with respect to the oocyte (angle ~75°) and gently lower the pipet until the tip penetrates the oocyte. The oocyte will at first dimple and then "spring back" as the pipet penetrates the membrane.

9. Inject the required volume of solution (see Note 7).

10. After all oocytes have been injected, place them into a glass vial containing approx 5 mL MBS and store at 17°C for about 30 min (see Note 5) to allow for intracellular distribution of the dye and caged IP3 prior to imaging Ca2+ responses.

3.1.4. Stripping the Vitelline Membrane for TIR Imaging

For TIRFM imaging, the plasma membrane of the oocyte must approach within less than 100 nm of a glass cover slip forming the base of the imaging chamber. It is therefore necessary to first remove the vitelline envelope, a connective tissue layer that remains even after collagenase treatment and removal of follicular cells.

1. After injecting collagenase-treated oocytes with calcium indicator dye, place a few oocytes in cooled hypertonic stripping solution at 10°C.

2. Wait for 10 to 20 min and then observe oocytes under a stereomicroscope using a fiberoptic illuminator with the light directed from the side. The oocyte should have shrunk, revealing the vitelline membrane as a surrounding translucent membrane. Grip the membrane using two pairs of fine forceps (Dumont 5), taking care not to pinch the oocyte, and gently tear open the membrane.

3. Use a dropper pipet to transfer the oocyte briefly to wash in a Petri dish filled with normal Ringer's solution, then transfer again to the imaging chamber. Once the vitelline membrane is removed, oocytes are extremely fragile and quickly adhere to clean glass and plastic surfaces.

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