Anti-SOX2 antibody [57CT23.3.4] (ab75485)

No staining in fixed frozen human tissue sections.

ANSWER:

Thank you for your call today and for letting us know about the trouble with these antibodies.

Please keep me updated about the results with these antibodies after trying the alterations that we discussed. Here is the article that I mentioned about performing heat retrieval on frozen tissue sections-

http://jhc.sagepub.com/content/53/11/1421.full

This article isn't specific for Brdu pre-treatment but it might be a helpful resource for you if you'd prefer to try a heat denaturation before using the HCl method. I've found another article (attached) which will useful for heat denaturation as well.

I look forward to hearing from you. Please let me know if you have any questions or if there is anything else that we can do for you.

   Dear technical support: This customer raised an inquiry about ab75485 (Anti-SOX2 antibody [57CT23.3.4]) and this customer used this antibody to conduct the IHC-P assay with human breast cancer tissue, and according to the datasheet, SOX2 should be detected in cell nucleus, but the image indicated the signal was detected in cytoplasma,  however this antibody is already out of our warranty period, therefore is there any suggestion can help this customer to improve his results I attached the IHC-P image and the IHC-P questionnaire in this letter, could you please help this customer to solve this problem. Thanks for your kindly assistance.   Best regards    

ANSWER:

Thank you very much for contacting us. I am sorry to hear the customer has obtained bad results with this antibody. I have checked our records, and I have not found an indication for a faulty batch or similar. I hope therefore that with the protocol tips we are able to resolve the problem. Please let us know however nevertheless the batch number and order date, so we can trace back the vial. In order to help to investigate the problem, I would like to to ask the following questions and make the following suggestions: 1.) I am not sure to what exactly the four images attached to the email do correspond to. Are the first two staining, and the second two the isotype control? 2.) To exclude that the observed staining is unspecific, I would strongly suggest to perform an isotype control. If there is any staining with the isotype controle, the protocol should be optimised until there is no unspecific binding anymore. If there is unspecific staining. I would recommend to double check the biotin and HRP blocking, as well as the general protein blocking. 2a.) To block endogeneous peroxidase activity, incubate the slides in 0.3% H2O2 in TBS for 15 min. 2b.) To block endogeneous biotin: either with milk and egg white, or a biotin blocking kit. 2c.) To block general unspecific protein -protein interaction, I can recommend to use 10% serum of the species of the secondary antibody as a blocking solution. Please recommend our online protcole as general guideline: http://www.abcam.com/index.html?pageconfig=resource&rid=11494#1 3.) I can also recommend to the customer to try to dilute the primary antibody further, for example 1/500, 1/1000 and 1/2000. This might decreases also unspecific staining. Please let me know if these suggestion help the customer or if the customer has questions or concerns. I am looking forward to hear back from you.