Anti-Fibroblast activation protein, alpha antibody (ab53066)

I stained with mouse tumor tissue. Tissue prepared by fixation method. 3days in 10% formalin and switch to 70% EtOH (Root temprature).

We use 5 different buffers.



1) TE9

2) CIT

3) Pepsin

4) Trypsin

5) No pretreatment



If you need further clarification, please let me know.

ANSWER:

Thank you for your reply.


With regard to the buffers, I was specifically referring to the blocking buffer, antibody diluents, and wash buffer.

I am concerned that you may be damaging the membrane resulting in high background or blocking your samples insufficiently. This is impossible to assess without more specific information regarding your protocol.


Regarding your tumor sample, do you expect a positive signal with the antibody? What are your positive and negative controls? Have you also run an isotype and no primary control to be sure your staining reagents are working properly?

Here is the summary of protocol:

1) Dewaxed

2) H2O2 15min

3) Antigen retrieval

1) TE9

2) CIT

3) Pepsin

4) Trypsin

5) No pretreatment

4) Blocking serum: NHS

5) Dilution: 1/100, 1/500 overnight

6) Rabbit impress method.



Expected results: We expected the cell membrain staining

Actual results: stained with nucleus

ANSWER:

Thank you for your reply with the protocol information.


What type of tissue are you staining (species and tissue)? How were the tissues prepared (fixation method, time, temperature)? What buffers are you using? Do they contain tween or triton? Is your no primary control clean?


Before considering a replacement, I want to be sure there are no modifications to the protocol that can be made to improve your results with this antibody.

Inquiry: I purchased antibody of FAP (ab-53066) Unfortunately, our experimental data shows that there is no specificity against FAP. Attached are the files representing our experiment. Due to the disappointing results, I'd like to get a full refund for the purchased product.

ANSWER:

Thank you for contacting Abcam regarding ab53066.


I am sorry that you have experienced difficulties with this antibody in IHC. I have reviewed the images that you have sent and I am hoping you would be able to provide some additional information. Would you please send me a detailed summary of your protocol and explain your actual results and expected results? I am not an expert in this area of study and thus I do not know the staining pattern you expect to see, the tissues you are examining, or the labels in your images.


I look forward to your reply so that I may assist you further. Please do not hesitate to contact me if you have any additional questions.

I am seeing three sets of bands with the most intense appearing between 37 and 50 kDa, and only a weak signal at the expected 90 kDa.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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.

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.

What was the positive control cells used in Flow Cytometry?

ANSWER:

Thank you for your enquiry.

This product was not tested in-house for Flow Cytometry but was used in the following publication.

Sromova L et al. Dipeptidyl peptidase-IV in synovial fluid and in synovial fluid mononuclear cells of patients with rheumatoid arthritis. Clin Chim Acta 411:1046-50 (2010). PubMed: 20361950

http://www.ncbi.nlm.nih.gov/pubmed/20361950?dopt=Abstract

According to the journal, fluid mononuclear cells (FMNC) was used. Since Abcam has limited access to journals, I could not confirm if any positive control cells were also used. However, I am sure you would be able to access the full publication from your university's library.

I hope this infomation will still be helpful.