[Histonet] von Willebrand factor IHC in rat kidneys

[Jan Shivers]

Antje,

I have also noticed decreased staining intensity with vWF in kidney
glomeruli, compared to other sites.  I use DAKO's product with Proteinase K
enzyme digestion for 5 minutes at room temperature, primary antibody at
1:600 for 30 minutes, LSAB2 detection system, AEC chromogen.

Jan Shivers
Univ. of Minnesota Veterinary Diagnostic Laboratory
USA

----- Original Message ----- 
From: <antje.marcantonio <@t> pharma.novartis.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, February 12, 2004 2:45 AM
Subject: [Histonet] von Willebrand factor IHC in rat kidneys


> Hello,
>
> I'm dealing with a strange phenomena.
> I have a nice working protocol for vWf in rat FFPE lung or heart,
including a 15 min pretreatment of trypsin, 1 hour incubation of primary
> antibody (Dako) 1:500
> followed by Envision/HRP. I get constantly strong and clean staining.
> The same protocol used for kidneys (yes, fixed and processed with the
> exactly same schedule) shows only  weak staining in the vessels and  very
> weak  signal in the glomeruli.
> I tried to use the primary more concentrated (up to 1:100) and to prolong
> the its incubation to 2 hours with only little improvement.
>
> Any ideas ? Should I consider another enzyme for pretreatment ? Longer
> digestion with trypsin simply increased the background ,not the signal.
> Is there any explanation why a protocol which works nicely in lung and
> heart is not applicable for the kidneys?
>
> Thanks to everybody,
>
> Antje Marcantonio
> Novartis Pharma AG
> BU Transplantation Research
> Basel, Switzerland
>
>
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