PCNA (proliferating cell nuclear antigen) staining?

[Rietdorf, Jens]

Dear list,

any experience with PCNA (proliferating cell nuclear antigen) staining
in human tissue culture cells?

A recommendation of a good antibody that is commercially available, or
any other advice strongly appreciated.

Thanks,

---
dr. jens rietdorf
head microscopy
novartis research foundation
friedrich-miescher-institute, wro1066.2.16
maulbeerstr.66, 4058 basel, switzerland
couriel:rietdorf(at)fmi(dot)ch

[Rietdorf, Jens]

TWIMC,

Another great success story for this list, the replies of Mariette Kemner-van de Corput, anh2006@, and in particular from Carol Norris and László G. Kömüves to my request have saved my users month of re-inventing wheels, frustrations etc. as you can realize from the wealth of information below.

Thanks a lot to all of you!

In brief, the anti-PCNA antibody from DAKO or Santa Cruz (clone PC10) is the standard reagent for PCNA detection and works well at least for non-fluorescent labeling of paraffin embedded fixed tissues or cell culture. This AB requires HIER in FFPE tissues. Alternatively, there is a rabbit monoclonal antibody against Ki67 (clone SP6, available from VECTOR) which works well both for IHC and IFL.
A methanol extraction step after PFA fixation may help to reduce a background of PCNA that is not involved in proliferation and is supposed to degrade in the presence of organic solvents. However, straight methanol fixation results in high levels of PCNA staining which is uniform across all phases of the cell cycle (visualized by PI staining), so to get cell-cycle specific staining, the cells have to be treated with 0.5% Triton before the methanol extraction. The nature of the large amounts of extractable PCNA label is unclear.
Useful references:  Larsen et al, Methods in Cell Biology vol 63 p. 419 (2001); Landberg and Roos, Cancer Res 51: 4570 (1991).



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rietdorf, Jens
Sent: Friday, January 16, 2009 15:31 PM
To: [hidden email]
Subject: PCNA (proliferating cell nuclear antigen) staining?

Dear list,

any experience with PCNA (proliferating cell nuclear antigen) staining
in human tissue culture cells?

A recommendation of a good antibody that is commercially available, or
any other advice strongly appreciated.

Thanks,

---
dr. jens rietdorf
head microscopy
novartis research foundation
friedrich-miescher-institute, wro1066.2.16
maulbeerstr.66, 4058 basel, switzerland
couriel:rietdorf(at)fmi(dot)ch

[M. van de corput]

you're very welcome :)

Mariette

____________________________________________

Dr. M.P.C. Kemner-van de Corput,
____________________________________________

MGC - Dept. of Cell Biology & Genetics
Erasmus Medical Center
Dr. Molewaterplein 50, 3015 GE Rotterdam
POB 2040, 3000 CA Rotterdam, The Netherlands

Office: H-Ee751; tel: [phone]
Lab: H-Ee710; tel lab: +31 10 704.3315
tel secr: +31 10 704.3169
____________________________________________

http://www2.eur.nl/fgg/ch1/cellbiology/
http://www.thesis.kemner.biz/
____________________________________________

Op Do, 22 januari, 2009 6:47 pm, schreef Rietdorf, Jens:

> TWIMC,
>
> Another great success story for this list, the replies of Mariette
> Kemner-van de Corput, anh2006@, and in particular from Carol Norris and
> László G. Kömüves to my request have saved my users month of re-inventing
> wheels, frustrations etc. as you can realize from the wealth of
> information below.
>
> Thanks a lot to all of you!
>
> In brief, the anti-PCNA antibody from DAKO or Santa Cruz (clone PC10) is
> the standard reagent for PCNA detection and works well at least for
> non-fluorescent labeling of paraffin embedded fixed tissues or cell
> culture. This AB requires HIER in FFPE tissues. Alternatively, there is a
> rabbit monoclonal antibody against Ki67 (clone SP6, available from VECTOR)
> which works well both for IHC and IFL.
> A methanol extraction step after PFA fixation may help to reduce a
> background of PCNA that is not involved in proliferation and is supposed
> to degrade in the presence of organic solvents. However, straight methanol
> fixation results in high levels of PCNA staining which is uniform across
> all phases of the cell cycle (visualized by PI staining), so to get
> cell-cycle specific staining, the cells have to be treated with 0.5%
> Triton before the methanol extraction. The nature of the large amounts of
> extractable PCNA label is unclear.
> Useful references:  Larsen et al, Methods in Cell Biology vol 63 p. 419
> (2001); Landberg and Roos, Cancer Res 51: 4570 (1991).
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Rietdorf, Jens
> Sent: Friday, January 16, 2009 15:31 PM
> To: [hidden email]
> Subject: PCNA (proliferating cell nuclear antigen) staining?
>
> Dear list,
>
> any experience with PCNA (proliferating cell nuclear antigen) staining
> in human tissue culture cells?
>
> A recommendation of a good antibody that is commercially available, or
> any other advice strongly appreciated.
>
> Thanks,
>
> ---
> dr. jens rietdorf
> head microscopy
> novartis research foundation
> friedrich-miescher-institute, wro1066.2.16
> maulbeerstr.66, 4058 basel, switzerland
> couriel:rietdorf(at)fmi(dot)ch
>