Anti-GAD65 antibody, prediluted (ab15299)

Customer has reported that she is seeing nuclear staining rather than cytoplasmic staining with these antibodies in IHC-P.

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with these antibodies. I saw that my colleague Charlotte previously made the following suggestion to you - "From the information that you have forwarded I suspect that the non specific binding may be due to the fact that you have not blocked endogenous tissue peroxidase activity. This is important where HRP/DAB system is being used as a detection system as endogenous peroixdases will interact with substrate and this may be resulting in a false positive signal. You could check this by conducting a no primary control, keeping the rest of your experimental protocol the same. If the same staining pattern is observed the non specific staining is a result of the secondary Ab and not the primary." Did you try using 1.6% H2O2 to suppress the endogenous peroxidase activity?

Thank you, and i look forward to hearing from you.

BATCH NUMBER 79029 ORDER NUMBER ????? DESCRIPTION OF THE PROBLEM Non-specific staining SAMPLE human ovaries PRIMARY ANTIBODY Ab Cam 7ml predil 30. min buffer after incubation DETECTION METHOD SA-HRP DAB POSITIVE AND NEGATIVE CONTROLS USED POSITIVE-HUMAN pancreas negative-human ovary/human pancreas ANTIBODY STORAGE CONDITIONS 4 FIXATION OF SAMPLE 10% NBF 10% NBF ANTIGEN RETRIEVAL Citrate buffer PERMEABILIZATION STEP ? BLOCKING CONDITIONS A/B 10 min. SECONDARY ANTIBODY biotinylated Ig anti-goat-mouse/anti-goat-rabbit HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 160sl HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? incubation different controls

ANSWER:

Thank you for your enquiry and you patience in awaiting a response. From the information that you have forwarded I suspect that the non specific binding may be due to the fact that you have not blocked endogenous tissue peroxidase activity. This is important where HRP/DAB system is being used as a detection system as endogenous peroixdases will interact with substrate and this may be resulting in a false positive signal. You could check this by conducting a no primary control, keeping the rest of your experimental protocol the same. If the same staining pattern is observed the non specific staining is a result of the secondary Ab and not the primary.

It is difficult to offer further advice form the information that has been forwarded, however a full protocol for IHC can be accessed via the “protocol” section of the on line data sheet. There is also a resource called “IHC World” which has specific protocols for IHC-P, along with information on specific blocking buffers.

http://www.ihcworld.com/_protocols/general_IHC/immunoenzyme_pod.htm

If you have any further questions then please do not hesitate to get back in touch with me.