I am doing immunohistochemistry of the c-Fos protein and I have a budget problem. For context, the experiment has been done over 3 years prior to my arrival and I have 40 brains done and 100 left. Is there a problem if I change the brand of the secondary antibody and the concentration of the primary antibody in the middle of the experiment? Do you think it could be a problem when it comes to publishing the results? The new secondary antibody I would use would also be anti-rabbit, but this would also mean that I would have to change the concentration of the primary antibody (anti c-fos) I used before.This new protocol would result in the same marking as I have now but I'm not sure I would be able to properly justify the changes.
Hello Marta,
I agree with Susanne Gebhard.
If you have to publish the data, it will make it harder for reviewers to establish the likely reliability of the results, and for researchers to reproduce the experiment, if you switch over to the new secondary antibody. All the 140 brain samples have to be treated in the same manner.
In the manuscript, you will have to mention the details of the antibody used such as source of the antibody, host species, code numbers, etc. If you don't, it will be difficult for reviewers to establish how well characterised the antibodies are and thus how reliable the data presented are likely to be. It also makes it more difficult for other researchers in your area of research to accurately reproduce the experiment. If you do mention the antibody details (old and new), you will still be questioned by the reviewers about reliability of the data.
If I were you, I would calculate the amount of antibodies required (both primary and secondary) for the total 140 samples after initial optimization steps, and order the antibodies of the same lot to complete the entire experiment. This will help to generate reliable data, and you will also avoid questions from the reviewers in this matter when you submit your manuscript.
Best.
I am also agree with Susanne Gebhard and appreciate the explanation by Malcolm Nobre.
Planning and optimization of any experiment is more important than performing the final experiments.
Good luck!
With regards,
Ishrat
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