Hi I made a stupid mistake. I stained tumoral cell suspension from mice tumor with human antibodies and Blok them with human antibodies then I used PBS/PFA 2%. any suggestion to correct this mistake? is that possible I wash the cell with PBS and then stained them again with the right antibodies.
I m really stressed and sad because I have been working on mice since 2 months then a stupid mistake.
thank you for your help
If I understand correctly, you used human antibodies instead of mouse antibodies to stain your tumor samples and then fixed them. I do not think you can strip the antibodies esp. with PBS. I would recommend that you take a little bit of your samples and run them in Flow.
1. If your human antibodies are cross reactive, they may have bound your antigens and you may see signals.
2. If they are not cross-reactive, then they may not be bound and your epitopes might still be free. If you do not see signals, then restain the rest of your samples with mouse antibodies and run them. Keep in mind that fix-stain does not work for all antigens.
The approaches mentioned above would help you salvage something from your situation. Regardless of which option works, you will have to repeat your study. It is unfortunate but necessary.
Hi Imene,
In other words - you are looking for antibody elution methods for cross-species reactivity (if there is any).
I do not know if any one of us can assist you. You have not mentioned the mouse tumor type, as per for example the Mouse Tumor Biology Database:
http://tumor.informatics.jax.org/mtbwi/index.do)
or the specific human anti-body you have used:
http://tumor.informatics.jax.org/mtbwi/live/www/html/antibodies.html
Even if you had supplied the necessary information however, your question would be best addressed by the manufacturer of the human antibody you have used.
One sensible approach would be:
First, please, read chapter 1 of this guide:
http://www.kanidis.gr/common/files/ANOSOISTOCHIMIA/DETECTION/ihc_staining_methods_5ed.pdf
Second, check with the antibody manufacturer:
- is cross-species reactivity a concern in your case
If yes:
- under what conditions is the antigen-antibody complex reversible (e.g. pH)
- what is the disassociation rate of the product you have used
- is disassociation usually complete or not
Third, ask the manufacturer for:
- any suitable flow cytometry elution methods.
Let us know how did it go :)
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining