If you are looking for an easy way to perform BrdU or PCNA staining, Zymed
offers complete kits for both PCNA and BrdU. The kits include a biotinylated
primary antibody (anti-BrdU/clone ZBU30 or anti-PCNA/clone PC10),
Streptavidin-HRP, DAB, positive control slides, as well as additional
ancillary reagents required for immunostaining. Because the primaries are
directly conjugated, non-specific binding of the secondary antibodies is
eliminated.

If you are interested in receiving a complete protocol please feel free to
contact me.

Best regards,

Aaron Levin
Senior Product Manager, International

Zymed Laboratories, Inc.
458 Carlton Court
South San Francisco, CA 94080
USA
tel:     650-871-4494 x107
fax:     650-871-4499
e-mail:  alevin@zymed.com
website: www.zymed.com

> -----Original Message-----
> From: tylee [mailto:tylee@itis.com]
> Sent: Tuesday, June 01, 1999 8:22 PM
> To: Fernando Capela e Silva; histonet@Pathology.swmed.edu
> Subject: Re: Cell Proliferation
>
>
> Fernando,
>
> First of all I assume you are working with an in vivo animal model.
>
> PCNA will be present and stainable in cells that are traversing the cell
> cycle. You may see different staining intensities in cells in different
> stages of the cell cycle, which is one of the disadvantages of PCNA. Cells
> not actively involved in the cell cycle should not be expressing
> detectable
> levels of this cyclin (i.e. PCNA).  The most common clone (?? PC-10 if I
> remember correctly) is species cross reactive.
>
> The BrdU method requires that you inject your animals with BrdU. This
> modified nucleoside gets into cells, gets converted to the triphosphate
> nucleotide form, then incorporated into DNA in those cells that are
> traversing the S-phase of the cell cycle. Thus, BrdU is specific for cells
> that are in the S-phase during the "labeling" period. BrdU-labeled DNA has
> to be rendered single stranded to achieve detection with all the
> monoclonal
> antibodies that I am aware of, thus some denaturation step is required.
> Denaturation is usually "acid treatment" which can result in a loss of
> morphology if you are looking for something else simultaneously. Another
> method is to use nuclease treatment which may not be as damaging to
> subsequent morphology. It is interesting to note that a group in Minn.
> (?perhaps Mayo) thought they identified a mAb (and I think
> published on it)
> that could recognize dsDNA labeled with BrdU; but, it turned out
> that their
> hybridoma was contaminated with mycoplasma that resulted in
> nuclease in the
> supernatant they were using as a source of mAb. I think this accidental
> discovery eventually led to them getting patent coverage on that rather
> lucky intellectual property.
>
> If you are going to isolate the chondrocytes, you might consider running
> flow cytometry instead.
>
> Ty Lee
>
> -----Original Message-----
> From: Fernando Capela e Silva <fcs@uevora.pt>
> To: histonet@Pathology.swmed.edu <histonet@Pathology.swmed.edu>
> Date: Tuesday, June 01, 1999 10:33 AM
> Subject: Cell Proliferation
>
>
> >What's the best way (and the vantages and disavantages)for the
> >quantification of cell proliferation (in my case chondrocytes): PCNA or
> >BrdU?
> >
> >Sincerely.
> >Fernando Capela e Silva
> >Departamento de Biologia
> >Universidade de Evora
> >Apartado 94
> >7002-554 Évora
> >PORTUGAL
> >
> >Telephone: +351-66-740 98 81
> >Telefax: +351-66-711 231
> >E-mail: fcs@uevora.pt
> >http://home.dbio.uevora.pt/~fcs/FCAPELAeSILVA.html
> >
> >
> >
>
>