Re: In vivo proliferation in vascular sections using Amersham BrdU ki t

From:Geoff McAuliffe

    First, how long between administration of BudR and death of the 
animal? Is it possible that  you are not allowing enough time for 
incorporation OR you are waiting so long that cell division has diluted 
the BudR label beyond the limits of detection?
    If your positive control is not working I think you should examine 
your experiment and technique before trying another kit or antibody. You 
may be doing something wrong. A good (better?) control is the epithelium 
of the small intestine, specifically at the base of the crypts between 
the villi. Always lots of proliferation there. I'm not up on cell 
turnover in spleen but I would expect some proliferation in white pulp, 
perhaps depending on the condition of the animal.
    BudR is considered (by some) to be more specific for proliferation 
than PCNA and if you can't make it work how will you make PCNA work?
    Have you talked to tech support at Amersham?


Shaposhnik, Zory wrote:

> Hello,
>I have tried to use the Amersham BrdU kit to look at proliferation in frozen mouse aortic sections and have not seen any staining with DAB. 5uM spleen sections were used as a positive control. No staining was seen there either.
>I confirmed that the Peroxidase linked secondary anti-mouse Ig 2a antibody
>Does anyone have any suggestions? Is a PCNA stain an easier way to look at
>proliferating cells in frozen sections? If so, what kit/technique would be
>Thank you,
>Zory Shaposhnik
>Division of Cardiology

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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