[Histonet] Re: Negative Control Tissue

[Gayle Callis]

I was taught that a true negative control should not be elimination of the 
primary antibody (NULL control) but use the immunoglobulin matching the host 
of the primary antibody and at the same concentration as the primary 
antibody.  Some people use nonimmune serum, but we prefer using IgG, IgG 
isotype, or IgM.  I also found out the hard way, in a research project, 
that using non immune normal serum was unacceptable to reviewers, and the 
whole, incredibly tedious and difficult immunogold staining had to be 
repeated before a manuscript was accepted.  A very hard lesson, and now 
there is a collection of IgG or isotype controls for all immunostaining 
projects available.

This immunoglobulin negative control can be used on a patient tissue.  The 
Ig negative control is there to evaluate nonspecific staining in that 
particular tissue.  If background occurs, then a source test may be 
necessary, and with elimination of a primary antibody, that is the first 
step in a background  source test.

There is an excellent discussion of negative controls,  tissue or reagent 
controls,  in the freebie, Dako's Education Guide, Immunohistochenisal 
staining methods, Fourth Edition and available as pdf on Dako Website. 
There are several ways of setting up a negative or positive control.  Their 
comment on page 114 of this manual, verbatim, "Finally, the diluent itself 
may be used as an alternative, which, however is neither efficient nor 
desirable" leads me to think the immunoglobulin control is more acceptable.

Gayle M. Callis
HTL/HT/MT(ASCP)
Bozeman MT 59715


-- Original Message ----- 
From: "Weems, Joyce" <JWeems <@t> sjha.org>
To: "Mike Pence" <mpence <@t> grhs.net>; "Sarah Kolekamp" 
<kolekams <@t> trinity-health.org>; <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, April 30, 2008 11:12 AM
Subject: RE: [Histonet] Re: Negative Control Tissue


This has always been confusing, but, if I understand correctly, the
negative control is the same patient tissue without primary antibody.
Use a negative for each detection system used if you have multiple
antibodies on the same block.

Joyce

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Mike
Pence
Sent: Wednesday, April 30, 2008 1:05 PM
To: Sarah Kolekamp; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Re: Negative Control Tissue

I think you would have to use the same tissue you use for your + control
to show that it "is working/staining" and "is not working/staining"
At least that is how we do it.

Mike

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Sarah
Kolekamp
Sent: Wednesday, April 30, 2008 11:06 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: Negative Control Tissue


I have a questions about negative control tissue, does anyone know what
the most universal tissue would be that we could use to act as our
negative control tissue.    We are looking for the tissue that could be
used with the widest array of antibodies.

Thanks!



Sarah Kolekamp HTL (ASCP)
Lead Histotechnologist
Saint Mary's Health Care

Phone Number (616) 752-6155
Fax Number (616) 774-0628

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