RE: [Histonet] negative immuno histochemistry control

From:"Luck, Greg D."

Hello all,

My approach is the essence of the negative control is to verify that serum
(and its components without the addition of an antibody) from the same host
species is not "on it's own" producing any non-specific background staining.
This being said would suggest to me that applying of any other reagent on
the negative slide except serum from the host species would not accomplish

Thanks, Greg

-----Original Message-----
From: Dawson, Glen [] 
Sent: Wednesday, March 08, 2006 6:25 AM
To: Edwards, R.E.; cynthia haynes;
Subject: RE: [Histonet] negative immuno histochemistry control

Although this is a good point, I don't think this is optimal unless the
normal serum or pre-immune that is used for the negative is from the exact
same animal/supernatant that the antibody is drawn from with the antibody
seperated out.  Throwing in something "else" is bringing another factor into
the equation.  How is a person to know that the "exceptable" negative was
collected correctly or that it is not contaminated.

That being said, I myself do the above to remain CAP compliant.  I used to
do the antibody diluent for whichever antibody I was using for the negative
control & I still think it's the most logical thing to do.  By using diluent
as the negative & keeping all other steps/reagents the same as the antibody
you are running, you are ensuring that it is, indeed, something in the
concentrated antibody that is causing staining not seen on the negative.  

Just My Opinion,

Glen Dawson  BS, HT & QIHC (ASCP)
IHC Manager
Milwaukee, WI

-----Original Message-----
[]On Behalf Of Edwards, R.E.
Sent: Wednesday, March 08, 2006 8:08 AM
To: cynthia haynes;
Subject: RE: [Histonet] negative immuno histochemistry control

For polyclonal antibodies use species specific(rabbit,goatetcetc) normal
serum, or  pre-immune if available: for  mouse monoclonals use the  specific
isotypic e.g.  IgG2a; simply omitting the  primary  antibody  or  replacing
it  with  diluent  alone  is  no  control.

-----Original Message-----
[]On Behalf Of cynthia
Sent: 08 March 2006 12:50
Subject: [Histonet] negative immunohistochemistry control

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

Cynthia Haynes H.T.

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