Anti-Leu + Met Enkephalin antibody [M09201] (ab6542)

Thank you very much for your offer. So, if it is possible, in exchange
for the refund on your enkephalin antibody, we would like to get a
calmodulin antibody instead - rabbit monoclonal (EP799y) anti
calmodulin antibody (ab45689) -, a 100 or 200 microliter barch, if
possible. We will try to order another anti human enkephalin antibody
from another source.
Thank you for your help in advance for exchanging these antibodies.
Best regards,

ANSWER:

Thank you for your reply. This case has been forwarded to me as my colleague is currently away from the office.

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 of alternative ab45689:

Order number 1075980
1 X vial ab45689

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.

I would like to ask you, what is the decision about the enkephalin
antibody, because the next human project will start at this friday.
It would be nice to know something about the process, because of the
urgent immunohistochemistry..
Thank you for your quick answer in advance,

ANSWER:

I am sorry for the delay in getting back to you.

I have been having some discussions with the source of this antibody and it seems that the epitope may be quite sensitive to the fixation. It seems that as you have achieved some staining using your rat samples that it is likely that it is not a problem of the antibody, but possibly of how the tissue is fixed. Although you have performed optimisation using differentantigen retrieval methods, it may be worth considering some different fixation methods also. For example, using acetone or methanol fixation of yourfrozen sections. Alternatively, it may be worth looking into the time used for fixation with formaldehyde (reducing the time).

If you would like to try an alternative vial of ab6542 I would be able to offer this, however, we do not have an alternative lot or an alternative antibody to the target to offer unfortunately. If you would prefer a refund or credit not this could also be arranged.

I hope this information has been of some help. I look forward to hearing how you would like to proceed.

Thank you for your really fast answer.
Please check, that you have the attached pdf with pictures in my
previous email?
The purchase order number was:
and the lot number was: GR72740.
We ordered your products through Izinta Kft. Hungary.
The scientific answers to your questions:
1. Human tissues were collected right after autopsy (by our swedish
coproduction partner). Frozen and criosectioned onto gelatin coated
slides.
These slides were fixed 5 min in 4% formaldehyde before immunohistochemistry.
Paraffin human sections were embedded of course according routine
paraffin embedding protocol.
These sections were rehydrated, numerous antigen retrieval methods
were tested with the same negative results (routine tripsin digestion
or 1% triton or microwave heating).
The positive results were obtained only with rat tissue, it was
transcardially perfused with aldehyde fixative, crioprotected in
sucrose, sectioned on criostat and mounted on gelatin coated slides.
Note, that negative results mentioned above could be seen only with
the enkephalin antibody. SP worked fine on paraffin slides and rat
samples too.
2. Dilution were (as i wrote in the attached protocol with pictures):
primary: 1:500, 1:250, 1:100 incubation 4 C or RT, overnight or one or
two days.
Secondary and peroxidase complex: 1:200 in all cases.
3. SP immunohistochemistry: the same secondary and peroxidsase complex
at the same dilution, same protocol at all.
Note: we use this protocol for years with a lot of antibody (i.e.
neuN, Calmodulin, importin, ChAT, etc.) with good results.
If the atteched pdf could not be opened, please write me and i send it again.
Thanky you for your work, best regards

ANSWER:

Thank you very much for providing that information.

I seems that the Enkephalin epitope may be sensitive to the how the tissue is processed. I am consulting with the lab to determine exactly how the IHC staining has been performed previously with this antibody to see if there are any suggestions which we may be able to make to improve the results observed to date. If we cannot find any suggestions to make, I will look into if we have an alternative lot which may be worth trying.

I will get back to you as soon as I can with this information. I am sorry for the delay and any inconvenience this causes you.

Dear Abcam!

We ordered anti enkephalin and anti substance P antibodies from you a
couple of months ago, and received them in february.
Since that time we are continuously working on the optimalization of
the protocol.
Substance P antibody worked fine, but the results of enkephalin
antibody was only a weak signal on the rat tissue with nothing in human.
The ordered antibody was designated to react in human too.
I attached the protocol and pictures of positive controls with SP and
Enkepahlin too.Details of the antibody is in the attachment.
The same protocol worked with your SP antibody fine, thus errors due
the protocol seems unlikely.
So we would like to ask you to solve our problem and send another
enkephalin antibody to be able to run our project.
Thank you in advance

ANSWER:

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

In order to understand the situation more fully, I was hoping you would be able to provide me with the following information:

1. How was the tissue collected? How was the fixation of the tissue performed?

2.In what diluent were theprimary and secondary antibodies diluted?

3. Was the same secondary antibody (and biotinylated-streptavidin-HRP complex) used as with the anti-Substance P antibody used?

4. I think I have located your order, could you just confirm that I have the correct information? Was the order number 1017456(Purchase order number xxxxx), and the ab6542 antibody lot number GR72740-1.

OnceI havereceive this information I will look at the protocol and see if there are any suggestionsI can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.