Re: [Histonet] negative immunohistochemistry control

From:"Margaryan, Naira"

I really do not understand all these discussion, sorry. May in clinic different than in research?

I always run negative control using a IgG isotype from the sheet in same concentration as a positive. But before to concentrate the antibody it is necessary to bring the protein concentration of negative control antibody to same protein concentration of your positive antibody. Run both, positive and negative side by side in same conditions. Only in this way you will be able to compare your staining.


This is a way how I work; proof my staining and learn,





Naira V. Margaryan, D.V.M., Ph.D.

Research Associate III

Children's Memorial Research Center

2300 Children's Plaza, Box 222

Chicago, IL 60614-3394

Tel: 773-755-6570/ext-8

Fax: 773-755-6594  


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From: [] On Behalf Of Chris Pomajzl

Sent: Wednesday, March 08, 2006 8:21 AM

To: HISTONET; Rene J Buesa

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 and

liver - if you do not run a negative control for that specific antibody, how

do you know if there is non-specific staining?


----- Original Message ----- 

From: "Rene J Buesa" 

To: "cynthia haynes" ;

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 reaction

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 how

many antibodies you run. The only requirement is that the negative slide has

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 could

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  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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