RE: [Histonet] IHC negative protocols

From:"Morken, Tim - Labvision"

Dawn, best practice is that you have one negative control for each antibody
type and method variation. What I have done to save slides is used pooled
mouse/rabbit normal sera for a negative, and use it at the highest antibody
concentration used in the run. 

However, negative controls are only useful if you see the same non-specific
staining on the "real" slide and the negative slide. If that is the case,
you many need to run several negative variations to rule out the cause. 

Of course, no clinical pathology lab uses the gold standard negative  for
commercial antibodies -- pre-immune serum from the same animal the antibody
came from in the first place.  It's completely impractical, but everything
else we do is only a poor approximation of that.

Tim Morken
Lab Vision - Neomarkers

Free webhosting for US State Histotechnology Societies:

-----Original Message-----
From: Dawn Cowie [] 
Sent: Wednesday, June 16, 2004 1:21 PM
To: histonet
Subject: [Histonet] IHC negative protocols

hello netters,

I have a question regarding negative protocols for IHC. CAP states that you
must use 1 negative control for each species of antibody, per each
pretreatment, per block. We have quite a large menu of antibodies that call
for different pretreatments plus the fact that some are rabbit, some mouse.
In order to comply with CAP we have 16 different negative protocols. From
different people that I have talked to, not too many are following this to
the letter. Is anyone else doing this? Is there an easier way? Are we taking
this too literally? Are we making this more difficult than we need to? Is
this too many questions? Any thoughts, ideas or suggestions would be greatly

Thanks in advance

Dawn Cowie HT

Pensacola Path
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