I have fixed xenopus laevis embryos with PFA and was in the middle of carrying out an immunostaining protocol but hadn't realised that I had run out of the secondary antibody that I needed.
I decided to wash the embryos 3x in PBS and leave in the PBS over the weekend. My question is: how long (if at all) can I leave between the primary and secondary antibodies? If I can, when I come to do the secondary antibody staining, do I need to reapply the primary antibody, or should I continue as normal with the secondary antibody as usual? Or do I need to start with fresh embryos?
Thank you!
Hi Julia!
This is also related to the expression level and stability of the antigen itself, as well as the quality of the primary antibody. Previously, we conducted experiments where the samples were soaked in PBS overnight after incubation with the primary antibody. For some antigens, this soaking had no impact, while for others, it did. However, soaking in PBS overnight may lead to increased background staining in subsequent steps.
If the tissue samples are particularly precious, you can try incubating the primary and secondary antibodies again. If the staining results are affected, it may still be necessary to obtain new samples and repeat the staining process.
Hope this helps u! Welcome to discuss any follow-up details.
Hi Julia, I'm glad this was helpful to you! If you have any further questions regarding details, feel free to reach out anytime.
I will also be sharing experimental protocols and key considerations on my account in the future. If you're interested, you're very welcome to check them out and share your feedback! If you have specific questions, you can also follow me and leave a comment.
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