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primary antibody not working?

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#1 entlein



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Posted 06 August 2009 - 05:50 AM

i am trying to make 3 different proteins visible in frozen sections (cryostat-slides) through immunohistochemical staining.
the sections fixed with 4%PFA and stored in sucrose-solution.
the protocol i've been working with:

1. fix tissue slides in 100% ice-cold acetone
2. wash with PBS
3. wash with 0,5%SDS in 1xPBS
4. incubate with 1st antibody for 2h at roomtemperature in blocking solution (Normal goat serum) with 0,3%triton x and pbs
6.wash in PBS
7.incubate with 2nd antibody for 1h
8.wash with PBS
9. microscopical examination for fluorescence

my 2nd antibody (alexa fluor) seems to be ok, because it worked with one of the primary antibodies for one of the 3 examined proteins.
i am suspicious that the other 2 primary antibodies may not be active anymore.
i also tried it without the pfa, and then got a weak signal, so i thought maybe the antigens are masked and i would have to demask the epitopes (e.g. citrate buffer), but am not sure if this makes sense with frozen sections or only works for paraffine-embedded tissue.
any suggestions what else could be the reason why the staining doesn't work? grateful for any suggestions!

#2 bob1


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Posted 06 August 2009 - 04:33 PM

I note you are doing a triple fix -cryosection, then PFA, then acetone... you should only need the section and fix in PFA or acetone shrinks cells so don't use it unless you have to.

You may need to use an antigen retrieval, though this is typically not needed for PFA fixed. It may be that the antibody is no good for IF/IHC - it might only work on denatured protein as in western blots.

Try doing primary overnight at 4 ˚C and permeablise in PBS with 0.5% triton X100.

Edited by bob1, 06 August 2009 - 04:34 PM.

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