I want to check if I can use GFP mice for IF quantification and look for proteins using secondaries that wouldn't excite GFP? (568 or 532)? Would GFP signal interfere with my staining and visualization process?
I'd recommend using a far-red fluorophore (e.g. AF647) for your immunofluorescence, if possible. You can use AF568, but you may have some signal spilling over from the EGFP depending on the microscope's filters.
However, the endogenous GFP signal is significantly diminished by alcohols. So if you are doing ethanol dehydration, the GFP signal may be low enough already to avoid significant background fluorescence.
Yes, it is doable using 2ries that less overlaping as suggested. But be aware that fixative can quench GFP but if its the eGGP shouldnt be a problem. And remember to do all of staining process away from the light not only after 2ry incubation not to lose GFP signal. Good luck!
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