Re: [Histonet] IHC run help

From:"Katri Tuomala"

Hi Meghan,

It sounds to me as if you have a blocking serum raised in an animal that is 
not compatible with the rest of the system. Make sure your blocking serum is 
not raised in the same species as either of your polyclonal primary 
antibodies (rabbit or goat). Your blocking serum should be raised in the 
same species as your respective biotinylated secondary antibodies.
For instance: when you are using rabbit polyclonal antibody, your secondary 
is most likely biotinylated goat-anti-rabbit, so the blocking serum should 
be normal goat serum. When you are using goat polyclonal antibody, the 
secondary would possibly be biotinylated rabbit-anti-goat and your blocking 
serum normal rabbit serum. If you were to switch the two blocking serums by 
mistake, you would get the reaction you are talking about.
If you are using kits, some of them don't tell you what the blocking serum 
is, so your best bet is to talk to the supplier, in case there has been a 
packaging error. Ask for another lot number...


Katri Tuomala
Hamilton, Ontario, Canada

----- Original Message ----- 
From: "Towers, Meghan [PRDUS]" 
Sent: Thursday, June 23, 2005 6:19 PM
Subject: [Histonet] IHC run help

> Ok, i have called several ihc labs about this problem i am having at my 
> lab
> and we are all stumped.
> all of our ihc runs are coming up positive when we put our dab on, even 
> our
> negatives.  here are some factors to consider in this problem that has us
> stumped.
> 1.  This procedure has worked for years and we have not changed a single
> reagent at all throughout the entire procedure.
> 2.  it's only happening with polyclonal antibodies (goat and rabbit) and 
> not
> monoclonal antibodies (mouse).
> 3.  since our mouse is coming out fine and our polyclonals are not, that
> means any reagents in common with the two clonalities (peroxide block,
> streptavidin, dab, etc.) are not the problem or else we would see the same
> reaction in the monoclonal runs, therefore, the problem must lie in the 
> only
> different steps between the two (blocking serum, primary, or secondary).
> 4.  since the negatives are coming out the same as the positives, the
> primary must no be the issue (plus, what are the chances of all our
> polyclonals going bad at the same time), yes, we've tried several 
> different
> polyclonal ab.s
> 5.  we have tried different tissue types cut from different sources at
> different times, as well as repeating exact runs we've done in the past 
> and
> they all turned brown, meaning it's not tissue or time specific.
> 6.  when we thought it was our blocking or secondary, we remade them both
> from an unopened, unexpired kit and still the same problem (even though it
> is the same kit we always use).
> 7.  we do our runs manually, not automatically, so mechanics being
> contaminated is not the problem.
> 8.  Please help us!  keep in mind while possibly pondering the problem, 
> it's
> always happening on the polyclonals, not the monoclonals, so if you think 
> of
> another reagent that is in common with the two, it's not or else it would
> happen on the monoclonal slides as well.
> 9.  the labeling we see, by the way, is completely nonspecific, like
> painting the slide brown, even the glass area around the tissue is turning
> slightly brown.
> if anyone has ever had this happen before or has any idea what it might 
> be,
> please let me know.
> thanks so much.
> Meghan A. Towers
> Johnson and Johnson
> Research Associate-Target Validation
> P.O. Box 776
> Welsh and McKean Rds.
> Spring House, PA 19477
> Phone: 215-628-5291
> Fax:  215-628-5966
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