Re: BrdU Staining

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From:"John C. Dennis" <>
To:Katie B <>
Date:Wed, 28 Apr 1999 15:19:50 -0500 (CDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

Katie B

Without knowing your protocol, here's my guess:

I'll bet your BrdU(-) cells (the ones that you'd expect to be BrdU(+))
contain the BrdU in regions of DNA that are not sufficiently denatured.
I've tried several different procedures and obtained varying results.
these include pepsin/HCl; NaOH; and DNase I procedures.  I've gotten the
best/most consistent results with pepsin/HCl.  

I'm assuming, by the way, that your antibody is specific for BrdU in
single stranded DNA.

You don't need to wait two hours because there is no BrdU available to the
tissues after 1 hr.  It's all in the liver by that time.

My animals also receive intrapartoneal injections.  I don't use forceps to
make the tent but my fingers instead.  Still, I do miss and end up with
subcutaneous injections.  In that case, the BrdU takes longer to enter the
blood stream but I don't know how much longer.

That may be a consideration but I'll still bet the more likely problem is
incomplete denaturation of the DNA when you apply the antibody.

Yours guessing

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849

On Wed, 28 Apr 1999, 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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