Re: [Histonet] Endothelial cell staining

From:Gayle Callis

First of all, you do NOT NEED ANY retrieval or digestion on Carnoys fixed 
tissue - there is NO formalin involved with this fixative - we had total 
success with DAKO Factor VIII, at 1:200 - 1:250.  Tissue was fCarnoys fixed 
overnight (we took out the chloroform since it is not a fixative, just 
there to remove fat/lipids).   Your retrieval may have ruined the antigen.

As for CD31 rat antimouse, we get no background staining with rat antimouse 
antibodies and we  use mainly BD Pharmingen antibodies.

It can be done two ways with either a biotinylated primary or CD31 
(dilution panel needed) come back with goat antiRat F(ab')2 biotinylated, 
ADSORBED TO MOUSE, and Strepavidin HRP.

For frozen section, 5 um air dry overnight, fix in 75ml acetone/25ml 
absolute ethanol mixture for 5 minutes at room temperature, DO NOT AIR DRY 
again,  go directly to buffer 3 changes.  Use appropriate rinses 
throughout, rinse buffer contains 0.05 - 0.005% Tween 20 and 0.2% goat 
serum.  Room temperature staining, no added temperature.  This fixative 
will give you good morphology and excellent staining.

Do DAKO peroxidase block (S2001) for frozen sections (for excessive 
endogenous Px, glucose oxidase method)
Normal serum block 10% goat/2.5% mouse serum mixture, 30 min
Strepavidin/biotin block (Vector), rather than avidin/biotin blocking
Primary antibody diluted in 5% goat- 30 min , negative control is either 
Rat IgG or isotype matched IgG
Secondary antibody diluted in NORMAL SERUM BLOCK with the mouse serum - we 
use Biosource/TAGO secondary, diluted 1:250 (0.5mg/ml stock) for 30 min
Strepavidin-HRP - Biosource, diluted 1:500 in buffer - 20  min
AEC+ from DAKO and control color development with a microscope on the 
positive control.

If you use biotinylated primary (negative control is Rat IgG-biotinylated 
from Jackson at exact concentration in ug/ml as primary)  just dilute this 
in the NORMAL SERUM BLOCK with mouse serum, 30 min -
Rinse and use Strepavidin-HRP or AP.

If you use DAB, you will have to adjust all antibody dilutions accordingly.

If you have too much endogenous peroxidase, there is a superior method to 
block peroxidase/pseudoperoxidase, called Glucose oxidase method.  I will 
be happy to attach via private email OR use an alkaline phos method 
instead.   DAKO has a new red chromogen for alk phos that is very 
sensitive, try that instead.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

At 08:28 AM 8/20/2004, you wrote:
>I just wanted to thank all histonet users for replying to my e-mail on 
>endothelial cell staining.
>To summarise my findings, von Willebrand Factor would work on Carnoy's 
>fixed normal mouse tissue but I could not get it to work on my tumour 
>sections. The tumour sections for some reason would not tolerate any 
>antigen retrieval even proteinase K for only 30 seconds would destroy the 
>tumour section. Maybe I didn't fix the tissue in the Carnoy's for long 
>enough. I got no staining with no antigen retrieval.
>I also tried the CD31 rat anti-mouse which is available from PharMingen. I 
>got this to work on frozen sections but with alot of background staining 
>which proved difficult to eliminate. Maybe my protocol was wrong or there 
>is alot of endogenous peroxidases in my tumours.
>I am especially grateful to John McGinley and Liz Chlipala who recommended 
>the CD31 from Santa Cruz which worked exceptionally well first time of 
>asking on formalin-fixed paraffin embedded tissue.
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