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Here are customer's answers:
1. I fully understand your concerns and it is highly unusual for 4 antibodies not to work for a customer. In this case, I can suggest to consider reviewing the sample preparation in particular. For example, try 1 fixing in 10% PFA for at least 24 hours.
The sample preparation is fine, and I have successfully stained these samples with multiple antibodies, some from abcam. These are rare samples of human tissue, and I cannot play around with the fixation.
2. Antigen retrieval can often require individual optimization for different antibodies with different epitopes. For example, I can recommend to try different time points for antigen retrieval. Try 2, 3, 5, 10 and 20 minutes.
I agree that antigen retrieval may be an issue. However, I did try two very different buffers, and two time points. Once again, I have a limited supply of samples, and cannot test all of these different time points.
3. As the target protein is nuclear, I can recommend to include a separate permeabilization step before staining.
I stained plenty of nuclear antigens on these same slides under the same conditions
4. I would suggest to try a lower concentration of antibody, 1:200 or even 1:500, to try and reduce the background.
Did that (1:500) - no improvement.
5. I can recommend to include 0.2% Tween in wash buffer to help keep the cells permeabilized and to wash away any non specific binding. Secondary antibody working well with other primary antibodies?
Secondary antibody works fine with other primaries. I don't normally have issues with non-specific binding.
6. 0.3% Triton is quite high, and may affect the staining. Try 0.2%.
As I wrote, I tried reducing and even eliminating triton, to no avail.
I have expended all of the sample slides available to me for finding the right conditions, and still have no results. This has cost great efforts and more resources than it should have. I will need to purchase a different antibody for this antigen from another company, so for now I guess I would like a credit note for a different antibody.
Thanks in advance foryour reply.
Asked on Aug 16 2012
Thank you for your message and for providing this further information.
I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments, and would be pleased to provide the credit note in compensation as requested, providing the product has been purchased within the 6 month guarantee period. In order to arrange this, I would appreciate if you could confirm your order number and date of purchase?
Thank you for your continued cooperation. I look forward to hearing from you with details of how you would like to proceed.
Answered on Aug 16 2012