Gonad Fixation

Reagents :

• 0.1% Tween PBS
• 3% paraformaldehyde
• Methanol (Stored at -20°C)

Materials:

• Dissecting Dish
• Drawn Pipette
• Eppendorf tubes
• Microcentrifuge

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Procedure:

1. With the drawn pipette, remove as much of the solution as possible. Be careful not to disturb the dissected worms.
2. Add 2 mL of 3% paraformaldehyde (PF) down the side of the dissection dish. Cover with parafilm, and leave in the dark for EXACTLY 1 HOUR.
3. Add 1 mL of 0.1% Tween PBS down the side of the dissection dish. Swirl the dish gently to get the gonads in the middle of the plate.
4. With a drawn pipette, gently collect the worms into 2 eppendorf tubes.
5. Spin gently 1000 rpm for 1 minute in centrifuge.
6. Wash with 0.01% Tween PBS.
7. Add 1 mL of MeOH. Place in -20°C for 5 minutes.
8. Wash 3x with 0.1% Tween PBS.
9. If necessary, proceed to Step 6 in the DAPI staining protocol.