I have been working on this for over one year. After I getting a lot of target cre-ert-gfp mice, I try to localize a group of cells expressing cre-ert-gfp by either immunofluorescence using GFP antibody or direct observation immediately after cryosection. However, I could not detect specific gfp signal except some autofluorescence by skin tissue. I can confirm that there are specific fluorescence when I observed mouse tail skin. However, after cryosection, it disappeared. Does anybody know the reason? I didn't use any chemicals to fix the tissue before cryosection.
Hi, Jifan.
Am I right that you are looking into hetero mice as you did not give any further info in the question. I believe that you are aware of specific tissues expressing GFP in hetrozygote individual mouse from the JAX home page and its related papers.The genome has only one copy of GFP gene fused to Cre, thus GFP-expressing cells driven by Foxj1 promoter have only small amount of GFP within the cells. If you are not doing proper fixation and proper permeation even in the cryostat sections, I doubt that cells show any decent GFP expression. You have to use thin section in cryo under confocal microscopy to intensify further with an anti-GFP antibody. With proper tissue processing, and use of both antibodies to GFP and Cre together may allow better staining to be detected only in the suggested tissues found in the reference.
Best wishes.
You can switch to WB analysis of your tissues. With WB anit-GFP antibody detects smaller fragments of GFP epitopes that damaged enough to be non-florescent anymore, it is also several folds more sensitive than confocal microscopy.
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