[Histonet] Carnoy's fixed tissue, Factor VIII Endothelial cell staining

[Gayle Callis]

Carnoys (you can leave out chloroform since it is not a fixative, there to 
remove fat only, safer to not handle chlroroform) fixed mouse tissue, 
overnight only, rinse 2x with 95% alcohol, and process starting at 100% 
(since this is alcolhol based fixative, you can start processing at higher 
concentration alcohol, clear and embed in paraffin.

DAKO, rabbit anti Human Factor VIII, von Willebrand factor, purified 1:200 
to 1:250 for 30 minutes,  Goat antirabbit or Donkey antiRAbbit-biotin - 
F(ab')2 frag of IgG from (Jackson).  Make sure your secondary  is adsorbed 
to mouse.

At beginning,

Endogenous peroxidase block,
Normal serum block 5 - 10% matched to host of your secondary 30 min.
Avidin/biotin block
Primary antibody (30 - 60 min) RT,
Secondary antibody-biotinylated approx 2 ug/ml, usually 1:250 for Donkey 
antirabbit biotin, from Jackson  30 min  dilute your secondary in 1 - 5% 
normal serum matching host of secondary.
Strepavidin-HRP (we do this in house dilution, Biosource/TAGO 5-6 mg/ml, 
diluted 1:500, for 20 min
Choice of chromogen, we prefer AEC+ from DAKO
Counterstain lightly with hematoxylin
Use AquaMount with AEC to mount coverslip.

Be sure to put 0.05% Tween 20 in all buffers and diluents.

I advise doing a dilution panel on your primary to reach optimal primary 
antibody concentration starting at 10ug/ml - work on either side of that 
and choose best staining.

You do not have to do any antigen retrieval with this method.

Good luck and let us know how your staining turns out, we had great success 
with this protocol on murine tumor looking for angiogenesis.

  09:18 AM 7/23/2004, you wrote:
>To whom it may concern,
>
>I'm a PhD student who is looking for advice on endothelial cell staining 
>in mouse tumour tissue for the purpose of microvessel density 
>staining.Most of the available antibodies don't work in mouse tissue. At 
>the moment I'm trying Carnoy's fixed tissue (fixed for four hours) with 
>rabbit anti-human Von Willebrand factor. Any suggestions would be greatly 
>appreciated.
>
>Jerry
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)