High background in phalloidin staining - (Dec/07/2009 )

Hello all, I encountered a problem in phalloidin to stain zebrafish embryos. there was high background, especially in the yolk granules, although we could see the staining on the cell surface. I do not know why.

this is the protocol used:
1. 4% PAF overnight
2. PBS 3x5 min
3. permeablize in PBS/0.1%TritonX100 for 10 min
4. PBS 3x5 min
5. blocking in 10% sheep serum for 1h
6. stain with 2.5 ug/ml Palloidin in blocking solution for 2hs
7. PBS 3x30min.
8. observation.

You probably need to permeabilise for longer, the embryos are large so it takes quite a bit of time for things to penetrate.

Yolks are full of protein, I am not sure that the yolk sac is made out of, but there is a good chance there is some actin there.

Doesnt formaldehyde give a lot green fluo background? And second the permeabilising. You could also add triton or tween to your washing solution. Note that this removes surface tension, so you may then want to do 1 wash with standard PBS before adding block or staining solution.

Have you tried other fixing methods, eg cryofixing or acetone?