Anti-PDX1 抗体 (ab47308)

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab47308 Anti-PDX1 antibody:
1. As you are performing a heat- induced antigen retrieval, an extra permeabilization step is not necessary for the FFPE.
2. Triton can be quite harsh on some epitopes, therefore, you may increase the clarity of the staining by reducing the Triton concentration during . Still, I can recommend keeping the Triton to your incubation solution to facilitate the antibodies solubility, but in a concentration of 0.025%.
3. As the necessary permeabilization step for the frozen sections, I would like to suggest to decrease the permeabilization step time with 0.1% Triton-X100in PBS down to 30 min.
4. To assure that the non-specific binding site are blocked, please block with at least 5% serum for 30 min before the antibody incubation.
5. The antibody concentration is very high compared to what we suggest on the datasheet, especially as the used tissue is the positive control. I would therefore suggest to increase the specificity of the antibody by decreasing the concentration down to at least 1:500 and to incubate overnight at 4C.
Hopefully, this will bring down the non-specific binding. Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.

Thank you for contacting us.

PDX1 will be expressed in the nucleus ofpancreatic islet and intercalated duct cellsin adult mice. I hope this helps, please let me know if you need any additional information.

 I have issued a free of charge replacement with the order number ****. This is for rabbit anti-PDX1, ab47267, to replace ab47308. The fixation is with 4% PFA for 5 minutes, followed by permeabilization with Triton X-100. The full protocol is below. Staining Cell cultures. 1. Rinse cells with PBS. 2. Fix in 4% paraformaldehyde in PBS for 5 minutes. 3. Wash 3 times with cold PBS for 5 minutes. 4. Permeablilize membrane with 0.2% Triton X for 10 minutes. 5. Wash twice with cold PBS for 5 minutes. 6. Block with 10% goat serum in PBS for 30 minutes. 7. Add primary antibody (1/5000) for 4 hours. 8. Wash 3 times for 10 minutes. 9. Add secondary goat anti-rabbit antibody (Alexa546) at 1/1000 for 1 hour. 10. Wash with PBS for 5 minutes. 11. wash with DAPI (1/10 000) for 10 minutes. 12. Wash with PBS for 10 minutes. 13. Mount with fluorescent mounting media. 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 assume you have the antibody ab47267, as that is the only rabbit anti-PDX1 in our catalogue that has been tested for ICC. We do not have samples of any of our antibodies but I can send a full vial as a replacement, perhaps from a lot different from what you have already tried. If the fresh vial gives a poor result again, we can send a different antibody if you like. Do you know the catalogue number of the rabbit PDX1 antibody that you have, and the lot number?

I am sorry to read that this is still not working for you. My guess is that the fixation is still too long at 2 hours, even with 4% vs. 10% PFA, or that there is lot-to-lot variability with regards to compatibility with formaldeyhe-based fixation. If you like, I will replace the antibody but I am concerned you will have the same result if I send ab47308. Are you interested in trying a different PDX1 antibody? We do not have another antibody raised in guinea pig but we have one from goat, ab47383, and another from rabbit, ab47267. If you do not want to try one of these (or any other primary antibody in our catalogue) free-of-charge, I can issue a credit or refund. I look forward to your reply.

Following up my previous message, the laboratory and I think the problem may be the 10% PFA fixation for 10 minutes at room temperature. The lab used 4% PFA and fixed for 2 hours at 4C. I think the fixation time could be made shorter than two hours, for instance 30 minutes at 4C. This will preserve the structure of the cell, but will hopefully minimize the cross-linkages introduced by the PFA that may be hindering antibody:antigen recognition. The acetone/methanol approach is an alternative, which does not create chemical modifications of the PDX1, but the laboratory has not tried this. Your current fixation protocol may be fine for other antibody stains, but each antibody:antigen interaction is unique, and ab47308 may have difficulty with 10% PFA at RT. Please let me know how the antibody works for you if you try one of these fixation modifications, and please contact me if you have any questions.