[Histonet] Re: Histonet Digest, Vol 111, Issue 32

[Tyrone Genade]

Hello,

On Thu, Feb 21, 2013 at 8:00 PM,
<histonet-request <@t> lists.utsouthwestern.edu> wrote:
> From: "Margaryan, Naira" <NMargaryan <@t> luriechildrens.org>

> I am trying to do  IHC on FFPE 4 weeks old zebrafish with different Abs. Is there a trick
> working with zebrafish? I am using the same IHC protocol I always use on human and
> mouse tissue and my Abs are  suppose to work on fish as well as human. I run human
> and fish sections together with same AR and IHC protocol. By the end I get beautiful
> staining on human section and or nothing or some
> fuzzy/fuggy/unclarified/undistinguished reaction.

I suggest you prepare some unfixed frozen sections for comparison. If
the antibody reacts to the unfixed tissue but not the fixed then you
probably need to perform one or another antigen retrieval method. I
have been having good success with
http://www.ncbi.nlm.nih.gov/pubmed/21603650

For my fish, Nothobranchius, I am now fixing for two days at 4 oC (in
PFA) and then decalcifying 15% EDTA (pH 7.3) for three days (the last
day in 30% sucrose dissolved into the 15% EDTA) before freezing in
liquid nitrogen for sectioning. This method is maintaining the
structure beautifully.

As regards using the above antigen retrieval method I have both soaked
the tissue (after fixing) in the 70 oC pH 9 TRIS and then decalcified
as well as decalcified, cryo-protected and sectioned (APTES coated
slides) and then performed the antigen retrieval at 70 oC. Both routes
work well for my fish and the sections don't detach.

If you cannot carefully dissect out the tissue of interest then I
suggest decalcifying the tissue. You only need one piece of bone to
catch on the blade and rip through the tissue to cause a lot of
frustration and waste a specimen.

I hope this helps.
-- 
Tyrone Genade Ph.D.
Department of Human Biology
University of Cape Town
South Africa

http://tgenade.freeshell.org
email: tgenade <@t> gmail.com
tel: +27-84-632-1925 (c)
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