I am trying to detect EYFP signals from Ifnbmob mice (made by Richard M. Locksley, Univ of California San Francisco-HHMI) tissues. These mice co-express Ifnb1 and EYFP from the Ifnb1 locus. The targeted gene co-translates EYFP, leaving cells that have made type 1 interferon-beta readily detectable by flow cytometry or immunohistochemistry. We have been trying using the recommended protocol but we are constantly getting weak signals. We even tried staining with GFP Ab. Does anybody have experience with this mouse system or something similar? We would appreciate your advice or suggestions.
Thank you in advance
https://www.jax.org/strain/010818
Hi Dacquin I'm afraid I have not had experience with these mice but I have used YFP or YFP fused proteins in the past and always found they were hard to detect (as some YFP - especially the older versions have a very low quantum yield).
The YFP in these mice also appears to be being produced from an IRES (so on the same RNA as the Ifnb1 gene) https://www.jax.org/strain/010818
The translation of protein from an IRES is always weaker from the gene under the up-stream (usually viral) promoter.
I just wanted to say be aware that alcohol fixatives (such as Methanol) can quench YFP/GFP fluorescence (so use PFA or formalin fixatives if fixing your cells).
Sorry I can't be more help!
Gary
I have no experience with YFP, but i do have experience with rfp and gfp reporter mice. Regarding flow cytometry the trick is to not fix your cells and measure them ASAP.
Thanks Gary and Anouk for your suggestions. I will consider them and try to improve the methods. I am also trying to block proteins secretion by using a golgi stop (Brefeldin A). Although it is very tricky to do so in in vivo settings, I want to give it try. Have you tried that before?
Hi Dacquin - only in cell lines I'm afraid. You might need to check with your ethical officer before giving Brefeldin to live mice though as it may be toxic.
Good luck with your work
Gary
Good for you Cs Ng. My setup requires stimulation with a tlr ligand. Have you detected positive yfp cells in murine lungs? Could you please share your protocol with me. For example, do you have to use an antibody? If yes which antibody do you use.
There is absolutely no chance that my method could be "crappy" as you put it. I can easily detect many other proteins in the same tissue (lung) using the same protocol.
Anyway thanks for your response and thanks already for sharing your protocol with me.
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