I have a FITC-labeled peptide that is supposed to bind only to certain gram-positive cells, but they seem to stain all the bacterial cells quite effectively no matter the buffer conditions. There is no free unreacted FITC present.
So do anyone know if the fluorescein (e.g. sodium fluorescein) molecule alone strongly interact with and label bacterial cells on its own?
The emission spectrum of fitc does overlap partly with autofluorescence of some cells. Does your visualisation show any fluorescence in non stained cells?
Paul Rutland the unstained cells do show significant autofluorescence in the green spectrum but are still distinguishable from the stained cells. I know that fluorescein diacetate diffuse across membranes and accumulate inside cells but fluorescein is supposed to be less hydrophobic / charged and likely not (?) interact much with the cell membrane despite being aromatic.
That is a pity Thomas Førland Oftedal but not entirely unexpected as you have tried different pH conditions. . Sorry that was all I had to offer. Hopefully others out there can help
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