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- Dissection dish
- Use 35mm or 60mm plastic culture dishes. Prepare Sylgard 184 elastomer (Dow Corning) by mixing base with the curing agent (10:1). Pour the mixture (about 3 ml for a 35mm dish) into a dish so that a thin layer (3-5 mm) of elastomer covers the bottom of the dish. Place the dishes under vacuum briefly to remove bubbles. Cure elastomer at 60°C overnight.
- Dissection pins by electrolytic etching:
- Prepare saturated sodium nitrite (NaNO2) solution in a jar with lid;
- Connect a fine clip to an electrical wire. Mount the clip on a wooden or plastic rod. Place the rod on a vertical-movement coarse micromanipulator (with the clip facing down). The clip will be used to hold the tungsten wire.
- Prepare a graphite rod (pencil lead will do) connecting to the wire.
- Connect the wire to a variable DC-AC converter so that the graphite rod will be the cathode and tungsten wire anode. Immerse the free end of the graphite rod in the solution.
- Cut ~ 5 cm of tungsten wire (.002" diameter wire is best for leech embryo); attach one end to the clip and the other end pointing down. Place the pool of saturated sodium nitrite solution directly below the tip of the wire.
- Apply ~10 volt DC. Lower the wire slowly until it touch the sodium nitrite solution. Bubbles should appear on the contact surface between the solution and wire. If you're getting small fireworks instead of bubbles, you probably wire it wrongly.
- Wait until bubbling stops (should take a minute or two). Raise the wire. Grab the free end with tweezers and cut the wire at ~0.5 cm from the tip. Place the electro-etched pin in the Sylgard dish filled with distilled water. Inspect the shape of the tip. If less than perfect, adjust the voltage and try it again until a desirable tip is obtained. Make a dozen or two. Bend the pins with tweezers and stick the sharp end into the Sylgard bed for storage.
- Dissection:
- Place the fixed (labeled or stained) embryo in a Sylgard dish filled with distilled water (if this is the last step before mounting) or buffered saline (if there are subsequent experimental steps after dissection).
- Pin the embryos down on its side. The pin should penetrate the yolk and hold down the embryo on Sylgard bed.
- Place a second pin at the anterior end of yolk mass and a third pin at the posterior end. Remove the first pin.
- Use the first pin to cut out the yolk mass and proboscis. Also, make several dorsoventral cuts in the provisional integument. Release the second and third pins after the yolk and proboscis are removed.
- Place the dissected body wall with a drop of water on a microscope slide.
- Drag the body wall to the edge of water drop; use surface tension to flatten and spread the body wall, with the ventral surface facing up and interior facing down.
- Blot off the water on the slide with bibulous paper. Avoid contacting the specimen directly with paper. Allow the specimen to air dry. Monitor the drying under a stereomicroscope. Do not allow the specimen dry up completely. Immediately before drying out add a drop of glycerol mount to the specimen (for the fluorescent dye labeled embryos, use antifading glycerol mount; for fluorescent protein labeled embryos, use PBS:glycerol 1:4) and place the coverslip. Seal the slide with nail polisher (use vacuum grease instead when using fluorescent protein).
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