[Histonet] IHC run help

Towers, Meghan [PRDUS] MTowers <@t> prdus.jnj.com
Thu Jun 23 17:19:43 CDT 2005

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
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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