nuclear staining without permeablization - (Aug/12/2008 )

Hey everyone,

I'm doing IICC for T47D cells. I'd like to see BP3 staining in the nucleus. The routine method always involves permeablization of nuclear membrane. That raises the argument the protein may leak out during the permeablization process. Is there any way I can get the fluorophore taged Ab into the nucleus without permeablizing the nuclear membrane?

Thanks!

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as far as I know Methanol fixes the cells as they are....Formaldehyde disrupts the lipid bilayer to some extent...but Methanol is ok..

We tried to stain nucleus once with Hematoxilin without fixation by Methanol, we couldn't get much blue stained nucleus, but when we fixed them prior to staining we got sharper blue color.

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Firstly, thanks . Just want to confirm, do you mean methanol will NOT permeablize nuclear membrane but only fix cells? BTW, I use formaldehyde and Triton-100 for fixation and permeablization step.

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I am convinced, that with fixation with formaldehyde, it does not matter if you permealize nuclear membrane, indeed, you have to to allow the antibody reach your protein. And formaldehyde crosslinks the proteins, so they stay were they are all the way after fixation.

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