[Histonet] negative immunohistochemistry control

Katri Tuomala katri <@t> cogeco.ca
Wed Mar 8 19:17:57 CST 2006

Almost in any given tissue (i.e. your positive control) has tissue 
components that do not and should not express the antigen you are staining 
for, so why run an extra slide for that? Doing the test slide by omitting 
the primary with the harshest retrieval, will give you all the information 
you need to show, that your procedure is working and if any non specific 
staining appears in your negative control slide, you would have to 
investigate further.


Katri Tuomala
Hamilton, Ontario, Canada
----- Original Message ----- 
From: "Andrea T. Hooper" <anh2006 <@t> med.cornell.edu>
To: "Jackie M O'Connor" <Jackie.O'Connor <@t> abbott.com>; "Histonet" 
<histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, March 08, 2006 1:12 PM
Subject: Re: [Histonet] negative immunohistochemistry control

Peptide inhibibtion is a good control to ensure
your antibody can be adsorbed by your antigen but
it does not tell you whether or not your antibody
will non-specifically bind to your tissue. It is
a good but not a sufficient control.

The best control is to stain tissue which DOES
NOT express your antigen, it should be negative.

>I'm not in a clinical setting - but I use peptide inhibition as a negative
>control for each antibody.
>"Chris Pomajzl" <cpomajzl <@t> cpllabs.com>
>Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
>03/08/2006 08:21 AM
>         To:     "HISTONET" <histonet <@t> lists.utsouthwestern.edu>, "Rene J 
> Buesa"
><rjbuesa <@t> yahoo.com>
>         cc:     (bcc: Jackie M O'Connor/LAKE/GPRD/ABBOTT)
>         Subject:        Re: [Histonet] negative immunohistochemistry 
> control
>I don't understand the idea of only one negative control per case, as
>opposed to each antibody. How does one evaluate non-specific staining of
>antibodies that do not have a negative control? What if non-specific
>staining is inherent to a certain antibody? For example, the new CAP
>question regarding staining of endogenous biotin, particularly in kidney
>liver - if you do not run a negative control for that specific antibody,
>do you know if there is non-specific staining?
>----- Original Message -----
>From: "Rene J Buesa" <rjbuesa <@t> yahoo.com>
>To: "cynthia haynes" <naje1972 <@t> yahoo.com>;
><Histonet <@t> lists.utsouthwestern.edu>
>Sent: Wednesday, March 08, 2006 7:51 AM
>Subject: Re: [Histonet] negative immunohistochemistry control
>>  Cynthia:
>>    The idea of the negative control is to try to determine if any
>detected in the case is due to specific or unspecific binding.
>>    You don't need to run a negative slide for each antibody you are going
>to test on the case, you only need a negative slide per case, no matter
>many antibodies you run. The only requirement is that the negative slide
>to come from the same tissue tested.
>>    What I used to do was to add buffer instead of the primary Ab and all
>the other reagents the same as in the slides run for specific Abs. You
>also add non-immune globuline instead of the antibody but in this case you
>would need 1 negative control for each type Ab (1 negative for monoclonal
>Abs and 1 for polyclonal Abs).
>>    I hope this will help you.
>>    René J.
>>  cynthia haynes <naje1972 <@t> yahoo.com> wrote:
>>    Good Morning everyone, I have a question about immuno
>>  staining. I've been away from this type of staining
>>  for a while and now I am doing them again on a regular
>>  basis. Why do you run a negative control with each
>>  run? Are you only suppose to put the normal serum on
>>  negative control only? I've forgotten; would someone
>>  please answer these questions for me. Thanks in
>>  advance.
>  > Cynthia Haynes H.T.
>  >

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