[Histonet] von Willebrand factor IHC in rat kidneys

[Patsy Ruegg]

Antje,
I too notice the different staining intensity for factor 8 in different
organs.  I do use proteinase K from DAKO for 5 min. instead of trypsin, you
might also try enhancing the DAB reaction with something like copper
sulfate.  Perhaps there is a difference of expression of the protein in
kidney cells.  I see the strongest F8 expression in lung.
Patsy

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of
antje.marcantonio <@t> pharma.novartis.com
Sent: Thursday, February 12, 2004 1:45 AM
To: histonet <@t> lists.utsouthwestern.edu
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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