Imaging

2.2.1. Confocal Imaging

1. Caged IP3 (Molecular Probes, Eugene, OR). Store at -20°C; light sensitive (but see Note 5).

2. Ca2+ indicator dyes (Molecular Probes). Store at -20°C; light sensitive.

3. Inverted confocal microscope with excitation laser (e.g., 488-nm argon ion laser).

4. 40x Oil objective (numerical aperture [NA] = 1.35) and low-viscosity immersion oil.

5. Oocyte recording chamber.

6. Glass cover slips (12-545C, 22 x 40-1, Fisher Scientific, Pittsburgh, PA).

7. Image acquisition and processing software. We use custom routines in Labview (National Instruments, Austin, TX) for acquisition and IDL (Interactive Data Language: RSI Inc., Boulder, CO) for processing.

2.2.2. Total Internal Reflection Fluorescence Microscopy

1. Stripping solution: 200 mM K-aspartate, 20 mM KCl, 10 mM MgCl2, 10 mM EGTA (ethyleneglycol bis-(2-aminoethylether)-N,N, N' N'-tetraaceticacid), 10 mM HEPES, pH 7.4.

2. High-Ca2+ Ringer's solution: 110 mM NaCl, 6 mM CaCl2, 2 mM KCl, 5 mM HEPES, pH 7.4.

3. Calcium indicator dye (Fluo-4 dextran; Molecular Probes, Eugene, OR).

4. Complementary RNA (cRNA) encoding N-type Ca2+ channel a1B-d and P3 subunits (mixed 1/1 to a final concentration of 0.1-1 |g/|L in diethylpyrocarbonate [DEPC] water).

5. Inverted microscope equipped for TIRFM, including TIRF (total internal reflection microscopy) objective lens (e.g., Olympus 60x oil immersion, NA 1.45), laser (e.g., 100-mW argon ion laser), low-light level, high-speed camera (e.g., Cascade; Roper Scientific, Tucson, AZ). See Subheading 3.2.8. for more details.

6. Low-fluorescence immersion oil for TIRFM (type DF; Cargill Laboratories, Cedar Grove, NJ).

7. Image acquisition and analysis package (e.g., MetaMorph, Universal Imaging Corp., West Chester, PA).

2.2.3. Two-Electrode Voltage Clamp

1. Two manual micromanipulators (e.g., model MM-3; Narishige USA, East Meadow, NY) mounted on the microscope stage.

2. Glass capillaries (e.g., 1.5 mm x 0.86 mm, 6 in., with filament; A-M Systems Inc., Carlsborg, WA).

3. 3M KCl solution for filling electrodes.

4. Chlorided silver wires (chloride silver wire by dipping in solution of household bleach for a few minutes) for electrodes and ground wire for recording chamber.

5. Voltage clamp (e.g., Geneclamp 500, Axon Instruments, Foster City, CA).

6. Recording and analysis software (e.g., pClamp, Axon Instruments; or Strathclyde WinWCP, available as a free download to academic users at http://www.bio-logic.fr/elec-trophysiology/winwcpandwinedr.html).

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