Re: BrdU Staining

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From:"Barry Rittman" <>
Date:Wed, 28 Apr 1999 13:34:15 -0500
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Most of your technique sounds reasonable to me and  50 mgs/kg is the
correct dose, but could you give us some more information please.

What are you using as the carrier vehicle?
What volume are you injecting?
What is the species?
Are you perfusing or immersion fixing?
What marker system are you using?

You really should not have a problem injecting IP if you gently raise
the skin and use a small needle on your syringe. If the problem is that
you are hitting some of the internal organs, try to gently shave the
area first to allow you to see more clearly.

The 48 hours fixation seems to be a bit longer than most labs would use.
Have you considered cutting the time down to 12-24 hours?

Take a piece of small intestine  through as a positive control.

Katie B wrote:

> 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:
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