These are iPSC derived human cortical organiods and I co-cultured them with tumor cells with GFP expression. After I fixed the tissues and amplified the GFP signal with an antibody. I would expect only tumor cells to be gfp+ but I also see GFP+ expression on my control cells. Could anyone have any explanation to that? I also checked them in microscope before fixation and there were GFP+ cells before I fixed them.
I meant before fixation, gfp+ cells in tumor and not in the control cells.
There are many situations in which paraformaldehyde fixation induces green cellular autofluorescence. Perhaps this is what you are experiencing. There are methods to suppress autofluorescence
https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.20392#:~:text=It%20is%20widely%20appreciated%20that,2%2C%206%2D9).
Article Characterizing and Diminishing Autofluorescence in Formalin-...
Article The effect of trypan blue treatment on autofluorescence of fixed cells
I had a bit of a similar experience and it might be that what you are seeing is just a background noise. I suggest for you to double check it with a different filter. If they are also visible with a different filter the it is just a background noise.
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