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Anti-PCNA antibody [PC10] - Proliferation Marker (ab29)

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If your product does not perform as described on this datasheet, we will refund or replace your product...

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This product is covered by the Abpromise guarantee. Our scientific support team are available to answer any questions or queries - fill out an inquiry form for ab29 for help.

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17 questions for ab29

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Question 1

Monday 14-May-2012

Thank you for your reply about antigen retrieval and PCNA. I am assuming that the 20 fix step you mentioned below would be for cut sections. We generally fix the specimen in whole mount then section and continue with our IHC without fixing the section. I study very small tissues (˜10mm blocks).
I have in the past fixed my tissues for 1 hour at room temp. in 4%PFA. But, have seen protocols for fixing up to 24 hours (I am assuming at 4C).

Under these conditions (1 hour fix), after cryosectioning, might I have to do antigen retrieval for PCNA? Also, do you recommend not fixing the specimen and only fixing the sections? I worry about tissue degradation--the OCT embedding takes a day (in sucrose…wont' tissues degrade int that time especially sitting in sugar?)

ANSWER:

  

Thank you for your reply.

The OCT embedding should not take a full day for 10mm blocks. You can soak the tissue in 30% sucrose / PBSfor 30 minutes at 4Cfor as many changes as are required for the tissue to sink (usually ˜3). Then the tissue can be moved to half OCT / half 30% sucrose for 15-30 minutes and finally into OCT for snap freezing in the block mold. Tissue degradation should not be an issue during this protocol, whether or not the samples are fixed. Once the sections are cut, they can be briefly fixed with 4% PFA or ice cold acetone or methanol.

While a longer fixation will be necessary to permeate the whole 10mm tissue block, after 20 minutes in a formaldehyde based fixative, the proteins will become cross-linked. The cross-linking can often obscure the epitope recognized by the antibody resulting in reduced signal. This is a significant issue for monoclonal antibodies that only recognize a single epitope, but less so for polyclonal antibodies. The cross-linking can be reversed by enzymatic or heat-mediated antigen retrieval.

If you are not observing any signal, I would definitely recommend either embedding the fresh tissue and performing a shorter fixation on the cut sections, or performing antigen retrieval. I hope this helps, please let me know if you need any additional information or assistance.

Question 2

Friday 27-April-2012

Do you recommend that we use heat mediated antigen retrieval on cryosectioned tissues it we are conducting IHC using Anti-PCNA antibody (PC10) -Proliferation Marker (ab29)? I understand this is common for paraffin sectioned tissues but have not heard of doing antigen retrieval on cryosectioned tissues.

ANSWER:

  

Thank you for contacting us.

This PCNA antibody gives good results on PFA fixed frozen sections without antigen retrieval when the fixation time is less than 20 minutes. For longer fixation, heat mediated antigen retrievalmay give a stronger signal. The attached reference describes how this can be performed on frozen or free floating sections. I hope this helps, please let me know if you need any additional information or assistance.

Question 3

Tuesday 24-January-2012

Yes, it has been very frustrating indeed. We chose the PCNA antibody because we wanted to use it as a control marker for proliferation, and at this point I'm thinking I want to try another proliferation marker that is similar to PCNA. I am thinking the Anti-Histone H3 (phospho S10) antibody - Mitosis Marker (ab5176) would be the next best thing. Would you be able to let us try out this antibody instead?

ANSWER:

  

I can send ab5176 if you like. I recommend having a look at some of the reviews and references to make sure this will be appropriate for your studies. If you have, please just confirm and I will send it out Tuesday.

Question 4

Monday 19-December-2011

Where is the epitope recognized by this antibody located on PCNA?

ANSWER:

  

Thank you for contacting us.

The epitope recognized by ab29, anti-PCNA, has been mapped to the amino acid range 111-125 of human PCNA.

Roos G et al. Analysis of the epitopes of proliferating cell nuclear antigen recognized by monoclonal antibodies. Lab Invest 68:204-10 (1993). PubMed: 7680082

Production of the PC10 clone is described in the following reference:

Waseem NH & Lane DP Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J Cell Sci 96 ( Pt 1):121-9 (1990). PubMed: 1695635.

Regarding an antibody that is reactive with the N terminus, we do not have one, but ab29 may be suitable for your studies depending on the sizes of the truncations.

Please do not hesitate to contact us if you need any more advice or information.

Question 5

Tuesday 11-October-2011

Yes, ******** the PO#. I think I'd like to go ahead and process the refund. I did try two of my aliquots through the process of my test stains and neither worked.

 

Thanks for your help.

 

ANSWER:

  

Once again please accept my apologies for you having issues with our ab29 antibody in your IHC-P. I have processed your refund as you requested.

If there is anything else I can do to be of assistance, please let me know.

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