[Histonet] Double labeling with antibodies that need different fixatives

[Adam .]

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
>
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>