Boureau Tristan wrote:
> Dear all,
> Of course GFP is a tool developped for living organisms studies, but
> I was still wondering if ever it could resist to some kind of
> treatment for fixation.
> Indeed I have GFP tagged bacteria, and I look their localisation with
> confocal microscope but I d like to couple this with some more
> classical fluorescence microscopy which needs making thin cuts of
> fixed tissues. So is there a method that would allow me to fix
> without destroying GFP.
> thanks in advance
> tristan
Hi Tristan,
I have no experience with GFP in bacteria, but in eukaryotic cells EGFP
can be fixed with (para)formaldehyde treatment. What we do is treat
cells with 3.5% paraformaldehyde in PBS for 15min at RT. This preserves
EGFP fluorescence quite well. However, it does not completely eliminate
the loss of EGFP from the cells when they are permeabilized for
subsequent immunofluorescence, most probably because only a cetrain
percentage of EGFP molecules are fixed. There is also an artifactual
"enrichment" of EGFP in the nucleus after fixation because the
cytoplasmic EGFP seems to be more prone to loss by diffusion.
Interestingly, this problem seems to be only for EGFP but not fusion
proteins with clear cellular localization, eg. nuclear. Those fusion
proteins are, at least in my hands, much less sensitive to experimental
loss. In any case, it is ESSENTIAL to check the localization of the
protein before fixation (ie. in live cells), to see if there was a
change in localization upon treatment.
NEVER fix EGFP expressing cells with organic solvents (EtOH, MetOH,
acetone....), because EGFP will leak out before it can fixed, so you´ll
have a total loss of fluorescence. Again, this is more pronounced for
EGFP alone than for fusion proteins.
Frank
