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Author Topic: advice: prestain live tissue with primary antibody  (Read 2888 times)

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Offline Achen

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advice: prestain live tissue with primary antibody
« on: March 20, 2013, 05:14:22 PM »
Dear IHC worlders,

Would appreciate your advice in a tricky situation.

My primary antibody will only bind unfixed tissue (something to do with conformational change on the target).

Would this work?

1) Incubate the live tissue with primary antibody for 2-3 hours with gentle agitation.
2) Wash
3) PFA fix and embed in paraffin
4) Section and detect with my secondary antibody

Would the PFA fix stop my secondary antibody from detecting the primary?

I understand that I would not be able to do antigen heat retrieval also.

Best regards, Antony


advice: prestain live tissue with primary antibody
« on: March 20, 2013, 05:14:22 PM »

Offline MT Scientist

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Re: advice: prestain live tissue with primary antibody
« Reply #1 on: March 21, 2013, 10:34:25 AM »
You may lose your signal upon processing/paraffin embedding.  If you don't lose the signal, I doubt that you would get much penetration of your antibody into the tissue interior, negating your original approach by pre-labeling.

Try frozen sections or antigen retrieval of the FFPE tissue.  An alternate antibody might also be available.

Offline Achen

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Re: advice: prestain live tissue with primary antibody
« Reply #2 on: March 22, 2013, 06:17:31 AM »
Thanks MT scientiest.

My tissue is 1mm thick needle liver biopsy and I was hoping the tissue would be thin enough for penetration. I have read that brain tissue staining is done on 1-3mm thick sections...

Yes, my biggest concern is that bound primary antibody would be cross-linked by the PFA fixative and "masked" therefore making it unrecognized by the secondary antibody.

I have tried antigen retrieval but the peptide/protein complex the antibody binds does not bind. I suspect the heat disassociates the peptide from the MHC-I, changing the conformational topology.

Offline gula

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Re: advice: prestain live tissue with primary antibody
« Reply #3 on: March 23, 2013, 03:03:24 PM »
I know, that in IHC there's postfixation after primary and before secondary for a better "affinity" of the binding.
This should lead to some crosslinking between primary and antigen and is used with primaries with low binding-strength.  I think, that polyclonal secondary in sufficient concentration can give a result, because it has the chance to find some unaffected epitops.
But this is done on sections, frozens or paraffin-embedded.

It sounds difficult to get a snapshot of the bound peptid. Perhaps something like native vibratome-sections and artifical, labeled peptid, with immediatly fixation? ...good luck.
gula

Re: advice: prestain live tissue with primary antibody
« Reply #3 on: March 23, 2013, 03:03:24 PM »