by Michael Koelle
modified from Beth Bucher and Andrew Chisholm
4/6/94
Phalloidin binds to filamentous actin. This stain allows visualization of mainly of muscles, but some other actin structures are visible.
1. Rinse worms off a plate with S basal, spin briefly in a clinical centrifuge, rinse once with more S. basal to remove bacteria.
2. Put 10 ul of worms in an eppendorf, freeze in liquid nitrogen, then immediately place in a speedvac to lyophilize the worms (takes ~5 min). Add 3-4 drops ice cold acetone, wait 5 min, remove as much of the acetone as possible and air dry/speedvac off the remaining acetone. Worms can be stored this way prior to staining.
3. Put 2 U fluorescein conjugated phalloidin (Molecular Probes, Inc. #F-432) in an eppendorf tube. Speedvac off the methanol to dryness. Add 20 ul "S mix" to dissolve the phalloidin, and add this to the dry worms.
S mix (1 ml)
743 ul dH20
250 ul 0.8 M Na phosphate pH 7.5 (recipe: 8.1 ml 1M Na2HPO4, 1.9 ml 1M NaH2PO4, 2.5 ml H20)
1 ul 1M MgCl2
4 ul 1% SDS
Let the worms stain at room temp (in the dark to prevent bleaching) for 0.5-1 hour.
4. Wash the worms 2X in 1 ml of PBBT (PBS + 0.5% BSA+0.5% Tween-20).
5. Resuspend the worms in ~20 ul 2 ug/ml DAPI in PBS (DAPI is kept frozen as a 1 mg/ml stock). Let the worms sit >5 min.
6. For viewing mix a few ul of worm suspension with an equal volume of mounting solution (90% glycerol, 10% PBS, 1 mg/ml phenlyenediamine).
7. Viewing: illuminating for DAPI fluorescence bleaches the FITC rapidly, so be careful.
