Hi,
My lab have GFP transgenic mouse.
GFP-transgenic mouse tissues 'Paraffin block'
I did IHC and I could detect gfp positive cell.
However, I tried GFP-transgenic mouse IF.
I could look GFP-positive only de-paraffin process. however,
My professor want to strong signal and co-immunostain.
So, I did deparaffin and then antigen retrieval and then 1% BSA blocking for 1 hour, 0.5% BSA incubation 1'nd antibody GFP(host:goat) at 4 ℃ o/n
and then 0.5% BSA incubation 2'nd antibody FITC(host:goat) 1hour at RT.
However, I could not detect any signal.
So, I tried change 2'nd antibody Cy3(host:goat) I could detect GFP-positive
signal.
So, I thought, FITC crush GFP signal?
I don't know why is this happen to me.
Please help me.
If you are using soft tissues, you can try frozen sections using PFA fixation followed by serial sucrose cryo-protection and O.C.T for embedding. During my GFP-mouse studies, I found that frozen sections tend to give better signal then paraffin embedded tissues.I see that you are using two goat antibodies- one after another. It would be better perhaps to use a rabbit anti-mouse antibody and a goat anti-mouse antibody. That way you can also mix the two primary antibodies as well as the secondary antibodies. Hope this helps and good luck with your experiments.
Sorry, a little hard to understand your question. Are you saying that you could detect the GFP with anti-goat Cy3 but not anti-goat FITC? I would suspect that the simplest explanation is that your FITC signal is weak. FITC can be a bit of a pain, and is very pH-sensitive.
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