Histonetters:

I have been using BrdU labeling of cells for quite
some time in my studies.  Lately, I've been having
problems with the staining quality of the tissues from
some animals.  I use BrdU from Sigma and anti-BrdU
from Becton Dickinson.  Tissues collected are fixed
with Zinc Formalin (Anatech) for a minimum of 48h,
processed together, and stained in large batches using
capillary gap immuno.  But about 10-15% of the animals
don't stain for BrdU at all, not even in the positive
control tissues (hair follicles, squamous epithelium).

It seems like the problem may be in the BrdU injection
itself.  I even increased the concentration to 60mg/kg
body weight for this last study, but still have this
erratic staining results.  (Our protocol calls for
50mg/kg.)  I know the injections are done properly
because I did them myself!

Injections were done by making a little "tent" of the
abdomen musculature, by pulling upwards with forceps,
while the animal is on their back, anesthestized with
4% halothaine.  This allowed me to inject into the
body cavity and not hit any organs.  If I had hit the
liver, would that account for the failure of the
incorporation of the BrdU into other tissues?  I then
wait 2h before sacrifice.

There just seems to be no pattern to this!  It's not
associated with any type of study exposure.  Does
anyone have any suggestions as to what else can
contribute to this?  I'm wondering if trying antigen
retrieval may help, but if the antigen isn't even
there, what next!?

===
Catherine "Katie" Bresee Bennett
Laboratory for Experimental Pathology
Department of Veterinary Pathology
Michigan State University

*new* e-mail: bresee98@yahoo.com

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