I am planning a multiplex ICC experiment. The issue is that several of my antibodies are anti-rabbit which would cross react with secondary antibodies. I was wondering if anyone has used antibody conjugation kits to preconjugate their primary antibodies to secondary fluorophores. I am worried that this process would impair the antibody function.
https://www.thermofisher.com/search/browse/category/iq/en/19501/IHC+&+ICC+Kits;jsessionid=B0E1C19ADBF4816D93BA092E73D3969E
Hi Tyler Panzner , is there any way you can get some of the antibodies from a different host? If another high-quality one i available, I HIGHLY suggest that route as it will save you a lot of time (and potentially money) in the long run! Otherwise, I've had success using sequential labeling as opposed to conjugation. The nice thing about sequential labeling is that you still give yourself some options with the secondaries and signal amplification, and you don't have to worry about conjugation efficacy, antibody function (as you mentioned) etc.
There's a few ways to do sequential labeling and Jackson Immuno outlines them really nicely (below) - I usually take the Option C route. Just be sure before you begin that all your antibodies are optimized solo before putting them together. Good luck!
https://www.jacksonimmuno.com/technical/products/protocols/double-labeling-same-species-primary
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