Raoxanne,
There have been several posts just now concerning proliferation and the "quirkiness" or "messiness" or "different intensities" with PCNA. Here is my experience.
PCNA is an antigen that is a nuclear, auxiliary molecule during proliferation. So in theory it is around during G1-S-G2-M when cell is proliferating. But cells spend most time in G0 (quiescent phase). Yet PCNA has a very long half-life and it certainly is present into G0 phase long after the proliferative, mitotic event. That is in literature and I have certainly seen that in cell lines and cells made to be "in synch" for their cell cycle. So you see some spectrum of PCNA intensity in what are actually non-proliferating cells, it can be quite true. Could be just left over. On the other hand the MIB nuclear protein, that a lot of people detect with the Ki-67 epitope antibody is relatively VERY short lived. It is there, and necessary, during proliferation, and as soon as that event is winding down in M phase, MIB is long gone. So it is "on and off" as opposed to on and then still there and then a little still there. Knowing the biology, and throwing into the mix fixation va
riance and section thickness variance and DNA repair possibilities and other things, I think it just turns out that MIB is easier to interpret and follow although PCNA, if you can read through the dirtiness, is still a good marker. For me, staining for MIB tracked very directly to gold standard BrdU staining and PCNA was also there but tough to interpret compared to BrdU. And I far prefer HRP to any alk phos in discrimination of interpretation of any of these but that is just a personal preference.
Ray Koelling
PhenoPath Laboratories
Seattle, WA
-------------- Original message --------------
From: godsgalnow@aol.com
> ANyone out there using PCNA?? I having been trying to work up this antibody and
> am not getting anywhere.? I will be using it in conjunction with?to MIB-1.?
> Since they are both proliferation markers I would expect them to look very
> similar, but they do not.? The PCNA seems to show different intensities in the
> staining where MIB-1 is either positive or negative.?
>
> I am using breast cancer and colon cancer as my controls.? I have tried using
> both an HRP and Alk Phos detection.? While the alk phos seems to be a little
> better than the HRP, it still isn't crisp.? I have the Biocare antibody and
> titered it out to 1:800 and I am using the Alk Phos Mach-4.
>
> Any suggestions?
>
> Roxanne
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