Dear Subrata,

First of all, it is unnecessary to do endogenous peroxidase blocking, since
you are using alkaline phosphatase labelled detection system. However I
don't think that is  what is giving you the background staining. Levamisole
in your chromogen solution is blocking the endogenous alkaline phosphatase.

How did you arrive at 1:50 dilution? Did you do antibody titre on known
positive tissue? Are you working on human or animal tissue? Is this antibody
a monoclonal raised in a mouse? Is the rabbit serum compatible  as a
blocking serum in rest of the method? Is your Envision a multilink or
binding only to a specific species? Lots of questions need answers before
any significant help could be offered. Your detection (Dako Envision) is
sensitive, so it is possible you need to titre your primary antibody a lot
further...

Let us know more details and I'm sure a lot of help will be offered...

Katri

Katri Tuomala
Department of Pathology
St.Joseph's Healthcare
Hamilton, Ontario, Canada


----- Original Message -----
From: "Subratab" 
To: "HistoNet Server" 
Sent: Saturday, June 14, 2003 11:39 AM
Subject: ED-1 IHC_ how to remove intense background staining


> Dear All,
> I am new in the field of immunohistochemistry. So my question may be
stupid one.
>
> To analyze renal macrophage infiltration I am using ED-1 Ab from Serotec.
My protocol is:
>
> 1. deparaffinization with xylene and graded alcohol
> 2. non-specific protein blocking by 5% normal rabbit serum
> 3. ED-1 Ab 1:50 dilution in 5% rabbit serum in PBS for overnight at 4 degr
C
> 4. blocking of endogenous peroxidase by 3% H2O2
> 5. Alkaline phosphatase-labelled polymer of Daco Envision system
> 6. Chromogen (buffer+fast red+levamisol) of Daco envision system
> 7. counterstaining with hematoxylen
> 8. dipping in ammonia water.
>
> But the slides showed intense red background staining with unexpected
number of positive cells.
>
> Please give me a suggestion? Do you think that primary Ab (ED-1) is too
much concentrated?
>
> Sincerely
> Dr Subrata Biswas
> PhD student
> Nephrology div
> UNICAMP, Sao Paulo, Brazil.
>
>