On 2016 Feb 01, Lu Wang commented:

Thanks for Cattoretti’s comment, including the literatures presented. Certainly, we agree with the information that specificity of any antibodies used in research should be standardized to ensure the accuracy of research (Bradbury A and Plückthun A). We also agree with the opinion that commercial antibody producers should test their antibodies more rigorously before selling them to scientists who often lack the resources and expertise to evaluate acquired antibodies (Holmseth S, et al). For example, no matter how to explain the result of a preadsorption test, it needs the enough free antigen to perform the test (Holmseth S, et al), hence PRDM1 antibody is also the case. As to the detection system of PRDM1 antibody in our study, please notice that both the final dilution and the secondary antibody were clearly stated in the paper. Meanwhile, cytoplasmic staining of PRDM1 is a question really puzzling us, because this expression pattern was different from all the previous reports. As you said that while cytoplasmic localization of PRDM1 needs to be proven and founded by some methods (for instance, preadsorption test, etc), however, our research design is scientific and rigorous. Firstly, we have established strict controls during beginning of IHC assay, including blank control. When the detecting system excluded PRDM1 antibody, no any signals were found in both cytoplasmic and nuclear of normal follicular epithelial cells. Secondly, we got same results in immunofluorescence detection. There is no exogenous or endogenous biotin participated in the response as everyone knows. Thirdly, we detected PRDM1 mRNA in whole cell mRNA, it exists in cytoplasms, too. So, we trust that there must be some unclear mechanisms to make this nuclear transcription factor expressed in cytoplasms. According the reasons mentioned above, we trust our results and decided to submit our research paper at last. Thanks again and best regards.

On 2015 Dec 04, Giorgio Cattoretti commented:

The claim that the cytoplasmic PRDM1 staining (a nuclear transcription factor) is indeed specific relies on the sole evidence of staining with an antibody known to produce non-specific cytoplasmic staining, whose concentration in the final dilution is not stated, counterstained with an unknown secondary antibody in a tissue known to be rich in endogenous biotin. As the sole first evidence of cytoplasmic localization of PRDM1, this is unproven, unfounded and false until proven otherwise with tools nowadays accepted for showing antibody specific staining in situ (see Bradbury A, Plückthun A (2015). Reproducibility: Standardize antibodies used in research. Nature 518:27-29. and Holmseth Set al (2012). Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity. J Histochem Cytochem 60:174-187.). Respectable Journals are increasingly called to enforce strict quality controls for these type of often flawed results. Best regards Cattoretti (see PMID:11232338, 12204275 and 15772984)

On 2015 Dec 04, Giorgio Cattoretti commented:

The claim that the cytoplasmic PRDM1 staining (a nuclear transcription factor) is indeed specific relies on the sole evidence of staining with an antibody known to produce non-specific cytoplasmic staining, whose concentration in the final dilution is not stated, counterstained with an unknown secondary antibody in a tissue known to be rich in endogenous biotin. As the sole first evidence of cytoplasmic localization of PRDM1, this is unproven, unfounded and false until proven otherwise with tools nowadays accepted for showing antibody specific staining in situ (see Bradbury A, Plückthun A (2015). Reproducibility: Standardize antibodies used in research. Nature 518:27-29. and Holmseth Set al (2012). Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity. J Histochem Cytochem 60:174-187.). Respectable Journals are increasingly called to enforce strict quality controls for these type of often flawed results. Best regards Cattoretti (see PMID:11232338, 12204275 and 15772984)

On 2016 Feb 01, Lu Wang commented:

Thanks for Cattoretti’s comment, including the literatures presented. Certainly, we agree with the information that specificity of any antibodies used in research should be standardized to ensure the accuracy of research (Bradbury A and Plückthun A). We also agree with the opinion that commercial antibody producers should test their antibodies more rigorously before selling them to scientists who often lack the resources and expertise to evaluate acquired antibodies (Holmseth S, et al). For example, no matter how to explain the result of a preadsorption test, it needs the enough free antigen to perform the test (Holmseth S, et al), hence PRDM1 antibody is also the case. As to the detection system of PRDM1 antibody in our study, please notice that both the final dilution and the secondary antibody were clearly stated in the paper. Meanwhile, cytoplasmic staining of PRDM1 is a question really puzzling us, because this expression pattern was different from all the previous reports. As you said that while cytoplasmic localization of PRDM1 needs to be proven and founded by some methods (for instance, preadsorption test, etc), however, our research design is scientific and rigorous. Firstly, we have established strict controls during beginning of IHC assay, including blank control. When the detecting system excluded PRDM1 antibody, no any signals were found in both cytoplasmic and nuclear of normal follicular epithelial cells. Secondly, we got same results in immunofluorescence detection. There is no exogenous or endogenous biotin participated in the response as everyone knows. Thirdly, we detected PRDM1 mRNA in whole cell mRNA, it exists in cytoplasms, too. So, we trust that there must be some unclear mechanisms to make this nuclear transcription factor expressed in cytoplasms. According the reasons mentioned above, we trust our results and decided to submit our research paper at last. Thanks again and best regards.