I'm trying to detect a specific protein in sheep tissues by IHC but the antibody against that protein was made in a sheep. What can I do?
Similar problems occur if you use antibodies generated in mice applied on mice tissue. there is a so called MOM (mouse on mouse) Kit from "vector labs". Basically the problem is solved/ diminished by blocking with mouse sera. usually 1-3%.
So you might use sheep sera to circumvent your problem. Start titrating the sera up to 3% (or even further); (obsverve the background you might get;. useappropiater controls as second antibody only)
Find an antiboby raised in a different species.
Where impossible to find, pre-incubate overnight 4°C your Ab with sheep plasma, try using it, cross fingers and check specificity in a very accurate way by western blotting. Good luck!
Thank you very for your answers. I need to use exactly that Ab, so I can't find it in a different species. I think in the same way that Matthew. Mathew, I'm going to use liver tissues and I usually use the ABC method but I don't know what's sAV seconadary. Could you give me more datails about this?. Thank you very much.
It is importan to do control sections: 1)positive and 2) negative
1) in a tissue section were the antigen is certainly present and 2) omitting secondary antibody
Thanks. I've read about this and it's possible. Ramos-Vara, 2005.. I'll try it!
Thanks. I've read about this and it's possible. Ramos-Vara, 2005.. I'll try it!
it will work, to reduce non specific results use blocking sera of normal healthy animal in which secondary antibody is raised. or use higher concentration of BSA or albumin fractionV. do not use universal secondary antibody it give huge negative results. keep thumb rule of keeping negative control i.e without pri antibody rest all experiences shared are fine.
Dear Veronica, you may try first blocking endogenous sheep IgGs using Rabbit anti-sheep IgG- IgGs or anti-sheep IgGs raised in some other species, which you will not be using as secondary antibody -Enzyme conjugate. After washing you may then add your specific antigen detecting antibodies raised in sheep. Then add anti-sheep-Enzyme conjugate. Use simultaneous negative control, where first endogenous sheep IgGs have not been blocked by rabbit anti-sheep IgG-IgGs (or whatever species you actually use).
Good luck.
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