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
>
>
>