Histonet, Katie and John,

I agree with John that the most likely cause of inconsistant results is
incomplete denaturation of DNA to allow access of the Anti-BrdU. There are
several hybridoma clones that are used by different companies; but, to my
knowledge, there are no Anti-BrdU clones that have the ability to recognize
Br-labeled DNA unless the DNA is denatured to single strands.  There were
some reports in the literature several years ago from Minnisota/Mayo
regarding a clone that did not require denaturation (and I think even a
patent application on the subject); however, it turned out that the
hybridoma clone was contaminated with mycoplasma and the nucleases from the
mycoplasma were denaturing the DNA.

The various denaturation procedures may have different effects on retention
of morphology... depending on the tissue type.  I have seen reported that
the acid treatments (i.e. HCl) are generally more effective at exposing the
halogenated-nucleotide antigen; but, if you are interested in seeing good
morphology or doing dual staining, there are published reports claiming
nuclease treatment gives the best results.

Ty Lee

-----Original Message-----
From: John C. Dennis <dennijc@vetmed.auburn.edu>
To: Katie B <bresee98@yahoo.com>
Cc: Histonet Server <histonet@Pathology.swmed.edu>
Date: Wednesday, April 28, 1999 4:09 PM
Subject: Re: BrdU Staining


>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: bresee98@yahoo.com
>>
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>
>
>