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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