I have transgenic reporter mice expressing mCherry and GFP endogenously. For a proof-of-principle experiment, I have to show their co-localization with other markers using immunofluorescent/IHC staining. What is the best way to fix and preserve the endogenous fluorescent signal? Or it is best to use fresh, unfixed tissue?
We fix tissues for a short time only, 10-20 minutes in 4% PFA, to preserve the GFP and tRed signals. You can cut frozen sections and fix these for 10 minutes. Alternatively, antibodies against GFP work very well on tissues that are fixed longer if you use antigen unmasking procedures.
We fix tissues for a short time only, 10-20 minutes in 4% PFA, to preserve the GFP and tRed signals. You can cut frozen sections and fix these for 10 minutes. Alternatively, antibodies against GFP work very well on tissues that are fixed longer if you use antigen unmasking procedures.
Thank you very much! I might have fixed the tissue too long in previous experiments (4h in 4% PFA)--the GFP signal seemed weaker than expeceted.
I agree with Gregory, you can even use 2 % formaldehyde. Try to avoid any organic solvent, because they reduce fluorescence signal. In addition, you can also use PLA (Proximity Ligaze Assay) to shown not only co-localization, but protein interaction.
It is easy and do not take too much additional time to compare with co-localization.
Good luck!
Great advice. I will keep them in mind while proceed along. Thank you~~
When I was analysing GFP signal in the skin of transgenic mice, I found that fixing even for 10 minutes in 4% PFA depleted the signal of the endogenous GFP. In my case I ended up carrying out my usual fixation procedure (10 minutes in 4%PFA for frozen sections or 2 hours for whole mounts) and used an anti-GFP antibody. I used a chicken GFP antibody from abcam (ab13970) and alexa 488 secondary. I have never worked with mCherry in a transgenic mouse model, but when I fixed human cells expressing mCherry with 4% PFA the signal was still quite strong. Just out of interest, which tissye are you looking at?
I am looking at vasculature of reproductive tissues, mainly in uterus and the ovary. In the previous experiment I fixed ovaries in 4% PFA for 4 hours, both mCherry and YFP signals are retained, but there seemed to have much fewer YFP positive cells than expected; I also noticed variable level of YFP signal in different cells, which was not observed when I was doing live tissue imaging of the same mice.
Thank you for the advice and discussion!
After receiving suggestions from you, I am trying different methods to see which works the best for our case. Shall know the answer soon.
When I have trouble with fluorescence being lost with fixation it is always helpful to have a look at the native fluorescence in live tissue, if that is a viable option. However, everything really depends on the level of expression of the fluorophores, and if they are both low you would probably be better off using antibody staining for amplification. The choice of mounting medium can have a big effect on stability of the fluorescent signal and can be fluorophore specific--what works for one may not work for another. You should try to find the best mounting media for your specific combination.
Thank you for another good tip. I was not thinking about mounting media before...
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