Hi Federica,

Can you give some more detail about what the problem is?

Do you get no staining or unspecific staining? How do you treat the the sections, before you stain them?

Best Simone

Hi Simone!

Thanks for your interest.  I’m new to this field!

To stain the sections, I usually use the following protocol:

-          Dissolve the paraffin by heating the slides at 65°C for 1-2h and then clear them with xylene.  

-          Rehydrate the sections with serial ethanol passages (100%; 95%; 85%; 60%; 30%) and then wash in dH2O.

-          Permeabilize with Nacitrate solution 10mM pH 6.

-          After cooling, incubate for 1h with phalloidin diluted 1:100 in blocking solution and then wash with PBS. (Once I used TritonX but I got the same result)

-          Incubate with dapi diluted 1:1000 in PBS1x.

-          After washing, mount the slides with mounting solution (fluoromount).

I suppose I get no staining, because all the section is fluorescent with any excitation filter (not only with those for phalloidin and dapi).

Best, 

Federica

Hi Federica,

I found the following thread on RG regarding phalloidine/DAPI staining on paraffin sections.

https://www.researchgate.net/post/Phalloidin_and_DAPI_staining_for_paraffin-embedded_tissue

There are a few good suggestions. The cytoskeleton ultrastructure could be lost due to insufficient fixation, which could explain the lack of Phalloidine staining.

If you are only doing the Phalloidine staining with a nuclear counterstain, the antigene retrieval step should not be necessary.

Have you tested the staining in cells/tissue which is not embedded? That might tell you, if your solutions are working properly and could give you an idea of what the staining looks like.

Good luck

Simone