[Histonet] Double labeling with antibodies that need different fixatives

[Andrea T. Hooper]

I haven't tried it directly but I don't see why not. If it's an amplification problem you can always amplify the fluorescence. That being said I am really not a huge fan of IF on FFPE especially when it comes to vessels as many vessels have high autofluorescence not to mention the autofluorescence from RBCs. I am afraid of false positives.


--- On Tue, 2/23/10, Adam . <anonwums1 <@t> gmail.com> wrote:


From: Adam . <anonwums1 <@t> gmail.com>
Subject: Re: [Histonet] Double labeling with antibodies that need different fixatives
To: "Andrea T. Hooper" <andreahooper <@t> rocketmail.com>
Cc: "Phebe Verbrugghe" <pverbrugghe <@t> meddent.uwa.edu.au>, histonet <@t> lists.utsouthwestern.edu
Date: Tuesday, February 23, 2010, 9:57 PM


On a related topic, does anyone know if the Biocare rat CD31 clone works for immunofluorescence on FFPE sections using a directly conjugated secondary antibody?

Thanks,
Adam


On Tue, Feb 23, 2010 at 1:06 PM, Andrea T. Hooper <andreahooper <@t> rocketmail.com> wrote:

Hi Phebe,
 
In my experience that CD31 clone you refer to doesn't work well mainly b/c of paraffin embedding -  in combination with PFA/formalin fixation. On PFA/formalin fixed frozen samples it works just fine with a trypsin antigen retrieval step. I find it's the paraffin that is the real killer. Try Biocare's anti-CD31, that works on FFPE. Or use another marker of endothelium (if that's what you are using CD31 for).
 
Alternatively, are you sure your second antibody only works in FFPE formalin fixed tissue? Because it may well work on formalin fixed frozen tissue, then you can use both on that instead (just remember to do the trypsin digestion). Or use fresh snap frozen tissue postfixed in PFA for 10 minutes then no antigen retrieval necessary.
 
Good luck!
Andrea Hooper


--- Phebe Verbrugghe <pverbrugghe <@t> meddent.uwa.edu.au> wrote:


        Hi all,
       
        I would like to do an immunofluorescent double labeling
with two
        antibodies but 1 antibody works on acetone fixed frozen
tissue but not
        on formalin fixed paraffin embedded tissue (CD31 BD
pharmingen 553370)
        and the other one works on formalin fixed paraffin
embedded tissue but
        not on acetone fixed frozen tissue. Is there any way I
could still do a
        double labeling and how?
        Also, does anyone have experience with zinc fixative? If
my antibody
        works on formalin fixed tissue is it likely to also work
on zinc fixed
        tissue?
       
        Thank you very much in advance,
       
        Phebe
       



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