Anti-GnRH antibody (ab8491)

ANTIBODY CODE ab8491 BATCH NUMBER 250 ORDER NUMBER 972A

DESCRIPTION OF THE PROBLEM Non-specific staining and high background

SAMPLE mouse brain

PRIMARY ANTIBODY abcam LHRH antibody/rabbit/from 1:200-1:50/overnight at 4C/ PBS 15min x 3

SECONDARY ANTIBODY Vector/biotinylated horse anti rabbit/1:200/2 hours/PBS 15min x 3 ABC (Vector) 1:100 1 hour

DETECTION METHOD DAB (sigma)

POSITIVE AND NEGATIVE CONTROLS USED positive: anothr rabbit Anti-GnRH (the same protein as LHRH) negtive: without primary antibody

ANTIBODY STORAGE CONDITIONS -20C

FIXATION OF SAMPLE 4% PFA fix 3 hours

ANTIGEN RETRIEVAL no

PERMEABILIZATION STEP 0.1% Triton X-100 30 min

BLOCKING CONDITIONS 1% milk powder in PBS and 1% Triton X-100 through out primary antibody incubation (4C O/N)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? different dilution, different individual animals different time

ADDITIONAL NOTES non-specific staining was high, but no specific staining. while another antibody (gift from others) can stain out very well.

Can I get some credit back for my next purchasing from you?

ANSWER:

Thank you for your enquiry and your detailed protocol. We are very sorry to hear that you are having problem with this antibody.

We have sold several vials of this antibody and do not have any problem with it.

According to the datasheet, this product has been tested and characterised on human. Other species have not tested so we do not have any information if the antibody works in mouse.

1. We would recommend using human brain section as positive control.

2. As we understand, you fixed the samples in 4% PFA fix 3 hours but have not applied any antigen retrieval step. Formalin fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross link proteins and therefore mask antigenic sites.

3. We would also recommend using 1% BSA as blocker rather than milk.

4. Your detection system is HRP-DAB. Have you tried to block endogenous peroxidase activity in the samples?

We hope that this will help to solve the problem.

Customer would like to know which epitope ab8491 recognizes.

ANSWER:

Unfortunately the epitope is not known. We know that it recognizes several epitopes on LH-RH. In the following reference there is data about the preparation of the antigen conjugate.

Van Rees, et al., 1984, Acta Endocrinologica 104: 272-278

To whom it may concern,

Can you provide me with any references that have used this particular LHRH antisera (ab8491). Of special importance to our work would be any referrences using immunocytochemistry on Frozen Tissue sections.

My concern about some of the technical details associated with the protocols listed is that this neuropeptide (LHRH) is quite small and typically such neuropeptides are easily extracted (i.e. removed) during parafin embedding processes. Thus, it comes as quite a surprise that this antisera actually works for immunocytochemistry on parafin embedded tissues.

Thanks.

Bob Thompson Univ of Michigan

ANSWER:

I have been unable to obtain a protocol for the use of this antibody in IHC from the originator. Furthermore there are no published references, so all the information we have is on the datasheet where we advise that use in IHC on PES should be without antigen retrieval and provide suggested started dilutions and a recommended dilution buffer. However, I can guarantee that if the antibody does not work as specified on the datasheet, we will provide an alternative product or offer a complete refund.