[Histonet] Double labeling with antibodies that need differentfixatives

Tue Feb 23 00:00:39 CST 2010

Hi Phebe

The first place to look when working up Antibodies is the Specification sheet from the manufacturer.  If you look at the BD sheet for their CD31 Immunohistochemistry is recommended for Zinc fixed paraffin sections and acetone fixed frozeb sections but not recommended for formalin fixed paraffin sections.

regards
Tony

Tony Reilly
Chief Scientist, Anatomical Pathology
Pathology Queensland-PA Laboratory
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>>> "Phebe Verbrugghe" <pverbrugghe <@t> meddent.uwa.edu.au> 23/02/2010 2:09 pm >>>
Hi Adam,

Thanks a lot, might just give it a go on just formalin fixed tissue
first.

Phebe

________________________________

From: Adam . [mailto:anonwums1 <@t> gmail.com] 
Sent: Tuesday, 23 February 2010 12:01 PM
To: Phebe Verbrugghe
Cc: histonet <@t> lists.utsouthwestern.edu 
Subject: Re: [Histonet] Double labeling with antibodies that need
different fixatives

I have no idea if it would work with regular formalin. In my experience,
many antigens work as well in zinc buffered formalin without any antigen
retrieval as regular formalin with antigen retrieval. But really, you
just have to try it yourself.

On antibodies I've gotten this to work, I use commercially available
zinc buffered formalin which comes in gallon jugs for around $50. We
don't do any special processing. We plop our sample (bones) in zinc
formalin overnight at 4C, decalcify in EDTA or formic acid (only
necessary for bones), and then embed just like any other sample.

Adam

On Mon, Feb 22, 2010 at 9:45 PM, Phebe Verbrugghe
<pverbrugghe <@t> meddent.uwa.edu.au> wrote:

Hello Adam,

Thank you very much for this very useful information!
Do you know whether this would also work on tissue fixed with
formalin instead of zinc buffered formalin by any chance? 
Also, could you give me the recipe for the zinc formalin and can
I use a standard tissue processor for embedding in paraffin or should I
use a specific protocol manually and if so, which? 

Thanks!

Phebe

________________________________

From: Adam . [mailto:anonwums1 <@t> gmail.com] 
Sent: Tuesday, 23 February 2010 10:52 AM
To: Phebe Verbrugghe
Cc: histonet <@t> lists.utsouthwestern.edu 
Subject: Re: [Histonet] Double labeling with antibodies that
need different fixatives

Although I haven't tried it myself, others have gotten CD31 from
BD to work on FFPE tissue using the tyramide amplification system on
zinc buffered formalin fixed sections. I've generally had good luck with
zinc buffered formalin myself for many antigens so it may work for your
other one.

See http://www.ncbi.nlm.nih.gov/pubmed/19052548 

Just to be clear, they used zinc buffered formalin, which isn't
the same thing as zinc fixative.

Adam

On Mon, Feb 22, 2010 at 8:41 PM, 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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