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**Industry Response**

It sounds to me like you are getting non-specific artifacts, though the
cause is unclear.

There are a couple other methods you could use.  One is a live-cell
plate assay that molecular Probes recently offers, called CYQUANT NF,
which utilizes a cell-permeant DNA-binding dye in combination with a
plasma membrane permeabilizing reagent. (The NF stands for no-freeze, an
improvement over the old CYQUANT products).

For fixed cells and microscopy, you could use BrdU (bromodeoxyuridine;
added to live cells, this compound gets incorporated into the DNA of any
dividing cells in that time frame), fix and perm the cells, then label
with an Alexa Fluor conjugated anti-BRDU antibody.  This is a
well-established method.

(I acknowledge association with Molecular Probes, which sells the above
suggested products and techniques)

Jason
Cell Biology
Molecular Probes / Invitrogen


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Phil Woost
Sent: Tuesday, May 23, 2006 1:58 PM
To: [log in to unmask]
Subject: PCNA Immunofluorescence

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We recently purchased anti-PCNA (and the corresponding blocking peptide)
for
use as a marker of cell proliferation. Mouse proximal tubule cells
(renal
epithelial cells) were grown on collagen-coated chamber slides or
semipermeable supports. Cells were fixed, treated with 10 mM citrate, pH
6,
(for antigen retrieval), permeabilized, and incubated with anti-PCNA
(1:100)
followed by an AlexaFluor-IgG conjugate. Immunofluorescence showed that
most
of the immunoreactive material (approximately 80%) was localized to the
cytosol, and only 10 - 20% was found in the nucleus. The blocking
peptide
effectively competed away about 80% of the immunofluorescence. --- The
expectation was that most of the immunoreactive material would be found
in
the nuclei of actively dividing cells and thus serve as a suitable
marker of
proliferation. Only a small amount, if any, was expected in the cytosol.
Can
you help explain my results? Alternatively, can you suggest another
marker
for cell proliferation?

Thanks,

Phil