[Histonet] head kidney issues

[Tyrone Genade]

Hello,

I am doing IHC on fish tissues, in particular the head kidney.

I am using DAB as chromogen. The tissue was fixed in Davidson's Fixative
and processed for wax. Sections were dried in an oven overnight and then
dewaxed and citrate was used for heat antigen retrieval. The sections were
then permeablized with PBS-Triton and incubated in methanol with H202 for
30 min followed by 20 minutes in pH 2.3 citrate (biochemically speaking it
should all be citric acid at this point) to denature any surviving
endogenous peroxidase. The last step may seem a bit extreme but I was still
getting signs of endogenous peroxidase activity in the head kidney sections
after the methanol and still am. Now I wonder if it is something else?

I found http://link.springer.com/article/10.1007/BF01003535 and saw this
http://www.ncbi.nlm.nih.gov/pubmed/16242344 and now wonder if the issue
isn't  5′-nucleotidase. The strong signal I am seeing has a granular
appearance (macrophages?). Has anyone had similar experiences and found a
solution? The former article implies using EDTA as an inhibitor.

Thanks
-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org
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