I am trying to stain stage 17 embryos using phalloidin without any success.
I am fixing the embryos in a 1:1 mixture of heptane/4% PFA after dechorionation in 50% bleach. I devitellinize by hand so I don't have to use methanol as it may interfere with the phalloidin staining. For the staining I use 1:500 phalloidin in PBST at room temperature.
So far I didn't have any success. None of the embryos are stained. I tried varying the Triton-X 100 concentration but this didn't seem to make any difference.
I also tried a heat fixation which seemed to improve the staining but destroyed the structures.
Does any one have a working protocol or any idea what I may have to change to get it working?
Before staining with phalloidin fixed embryos for 30 min at room temperature in a 1:1 mix of heptane and 8% formaldehyde.
After fixation, hand-devitellinize embryos in PBS, block in 1% BSA/0.1% Triton in PBS for 30 min, incubate in 1 µgml−1 Alexa594–phalloidin for 30 min, wash three times for 15 min in PBS containing 0.1% Triton and analyse on the confocal microscope.
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