Anti-Oct4 antibody - ChIP Grade (ab19857)

I would very much like to get hold of your recommended protocol forimmunohistochemistry ofparaffin and frozen sections. Our technicians at the university are very experienced atimmunohistochemistry and they have tried several different protocols to make the antibody work - but nonspecific uptake has been a problem. We will compare our protocols with yours.

Thank you also for your kind offer to replace the Oct 4 antibody that we have. Would it be possible to have this antibody: Anti-Oct4 antibody (ab18976).

Given how extensive our efforts have been to makeab19857 work with tissue sections, it would be nice to try with an alternativeabcamOct4 antibody.

I look forward to seeing your protocol and hearing your opinion.

All the best

ANSWER:

I have created a new order for you, the order number is 1077837 with antibody ab18976 as per your request.

You can check the protocol on our website. These are

http://www.abcam.com/index.html?pageconfig=resource&rid=11383

http://www.abcam.com/index.html?pageconfig=resource&rid=11384

Please not the main difference between IHC-Fr and IHC-P is fixation of the tissues and sections.

I hope this information will be helpful. Should you have any other inquiry please do not hesitate to contact me.

I have an enquiry regarding your products; ab20650 and ab19857.
I would like to confirm with you that ab20650 can be used to elute
Oct4 proteins immunoprecipitated by ab19857.
I would be most grateful if you could allow me yo know
whether it is possible.

ANSWER:

Thank you for contacting us.

The peptide ab20650 has not been specifically validated for use eluting proteins that were immunoprecipitated using ab19857, but it should be suitable for this purpose when used at a high enough concentration. I would recommend allowing the column to drain completely and elutingthe bound protein with five one-column volume aliquots of a solution containing 100ug / mL peptide in TBS. I hope this helps, please let me know if you have any additional questions.

IHC-P
- How long the tissue sections were fixed in formalin? Over 24 hours
- What is the antigen retrieval buffer e.g. citrate (pH 6) toTrisEDTA pH (9.0). Sodium citrate pH 6
- For how long antigen retrieval was done e.g. 10 minutes boiling or 20 minutes boiling etc.1min40seconds at 125 degreesCelsius in anMenorini antigen retrieval unit
IHC-FR
- What was the fixative? None
- What was the fixation time? No fixation
Thanks and regards,

ANSWER:

According to our records, ab19857 was proving difficult to use inIHC and we were in contact in order to help resolve the issue.

Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the difficulties experienced. If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution.

If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records.

We wish you the best of luck with your research and look forward to a reply.

I have purchased this antibody a few times previously and it has always performed well in my IHC experiments. But this new vial I received is just giving background staining on the same samples and using the same protocol I have used successfully in the past.

ANSWER:

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab19857.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

1. With reference to anti mouse FSHR antibody (ab115633)- works only for Western but not for immuno-localization studies. In fact two bands are observed by Western. Pl see attached results. I actuallyhadordered for immuno-localization.

2. The OCT-4 antibody which was supplied as replacement (lo no. GR56247-1) ... has again not worked during immunolocalization. We had ordered for 5 vials.. 2 vials were sent as replacement but no results of IHC. In fact we used a new detection kit and also used another antibody - just to confirm our system. Everything is fine - but OCT-4 immunolocalization is just not working as the earlier lot for Ab (our purchase order 617288 dated 31.12.09). But it works fine on Western using embryonic stem cells. So I do not understand where the problem is. But our 3-4 papers are pending because we need a good polyclonal antibody from ABCAM.

Please let us know

ANSWER:

Thank you for letting me know of your progress in using anti-Oct4 antibody (ab19857) and anti-FSHR antibody (ab115633).

I am sorry to hear that these antibodies are still not performing as you had hoped. In regards to anti-FSHR antibody (ab115633), as discussed previously, to our knowledge this antibody has not been used successfully in performing IHC staining. This does not mean that it would not work, simply that we have no reports of it and that considerable optimisation may be required in order to obtain good staining. As the antibody is working in Western blotting, I would suggest that either the antibody is not capable of detecting the epitope as presented in fixed tissue, or the epitope has not been sufficiently revealed. Some optimisation, particularly with the antigen retrieval may be benificial. Unfortunately, we do not currently have any antibodies in our catalogue which can be guaranteed to work with IHC staining of mouse tissue.

I am glad that you have now been obtaining good western blotting results with the anti-oct4 antibody (ab19857). Could you explain a little further the unexpected results you have been obtaining when using this antibody for IHC? Are the results the same as before (non-specific staining) or are you seeing no staining at all?

In order to understand exactly the protocol you are currently using and the results obtained, could you please fill out the questionnaire attached to this email. I realise that you have filled this in previously but I just want to understand which parts of the protocol have been amended and if you could also include images of the results obtained (including the western blot) that would be very helpful.

I hope this information has been of help. I look forward to receiving your reply.